CN106163532B

CN106163532B - Compositions characterized by attenuated newcastle disease virus and methods of use for treating neoplasia

Info

Publication number: CN106163532B
Application number: CN201480048630.4A
Authority: CN
Inventors: 程星; D.卡罗尔; M.麦库尔特; M.贾林斯基; 金红
Original assignee: Immune Medical Co Ltd
Current assignee: Immune Medical Co Ltd
Priority date: 2013-09-03
Filing date: 2014-09-02
Publication date: 2019-12-31
Anticipated expiration: 2034-09-02
Also published as: AU2014317215A8; PT3041490T; SI3041490T1; DK3508209T3; CA2922071A1; US20160208222A1; CA2922071C; LT3041490T; AU2019204419A1; KR102310692B1; TWI653334B; KR20210048605A; EP3041490A1; ES2708755T3; KR102285894B1; PT3508209T; WO2015032755A1; PL3041490T3; CY1121993T1; SI3508209T1

Abstract

The present invention provides methods for inducing tumor regression in a human subject using a modified mesogenic strain of Newcastle Disease Virus (NDV) having a modified F protein cleavage site that is non-pathogenic (lentogenic) to poultry but exhibits oncolytic properties. The disclosed methods provide a safe, effective and reliable means of inducing tumor regression in an individual in need thereof. These methods overcome the disadvantages of using pathogenic strains of viruses for human therapy.

Description

Compositions characterized by attenuated newcastle disease virus and methods of use for treating neoplasia

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application serial No. 61/873,039 filed on 3/9/2013, the contents of which are incorporated herein by reference.

Background

Newcastle Disease Virus (NDV) is an infectious avian disease caused by avian viruses, affecting many domesticated and wild birds. Human exposure to infected birds (e.g., in poultry processing plants) can cause mild conjunctivitis and flu-like symptoms, but NDV does not otherwise pose any harm to human health, and most people are seronegative for NDV. NDV pathogenicity, as determined by the intracerebral pathogenicity index (CPI), is classified as high (velogenic), moderate (mesogenic) or low (hypotoxic) based on viral pathogenicity in chickens. Due to agricultural considerations, since 2008, moderately and strongly virulent NDVs with chicken virulence (ICPI >0.7) have been classified by the USDA as "selective agents" which list of toxins includes biological agents that have the potential to pose serious threats to human and animal health, to plant health, or to animal and plant products.

Naturally occurring forms of NDV have been used in clinical studies as biologicals for immunotherapy and viral therapy. Because of the ability of viruses to selectively kill human tumor cells, with limited toxicity to normal cells, NDV has shown promise as an anti-cancer agent. However, the development of NDV as an anti-cancer agent has not progressed as NDV is reclassified as a selective agent. Other oncolytic viruses have shown considerable promise in clinical trials. To facilitate the development of NDV as a cancer therapy, new forms of virus are needed. Ideally, such new forms would still retain their ability to target tumor cells, but would no longer cause disease in birds.

Summary of The Invention

As described below, the invention features compositions and methods for treating neoplasia.

In one aspect, the present invention generally provides an attenuated Newcastle Disease Virus (NDV) having an F protein cleavage site of glycoprotein b (gb) of the NDV LaSota strain or of Cytomegalovirus (CMV) (S116). In one embodiment of the invention, the modified F Protein Cleavage Sequence (FPCS) has one of the following sequence modifications, S116:¹¹¹H-N-R-T-K-S/F¹¹⁷；S116K：¹¹¹H-N-K-T-K-S/F¹¹⁷；S116M：¹¹¹H-N-R-M-K-S/F¹¹⁷；S116KM：¹¹¹H-N-K-M-K-S/F-I¹¹⁸(ii) a Or R116:¹¹¹H-N-R-T-K-R/F-I¹¹⁸. In another embodiment, the attenuated virus strain is a modified 73T virus strain. In yet another embodiment, the attenuated NDV virus is a R73T-R116 virus. In other embodiments, the virus has an increased HN-L intergenic region. In yet other embodiments, the HN-L intergenic region is a non-coding sequence that is between at least about 50-300 amino acids in length. In other embodiments, the non-coding sequence is derived from paramyxovirus type 1 (APMV-1), Respiratory Syncytial Virus (RSV), or random sequences. In yet another embodiment, the non-coding sequence between HN and L genes is 60, 102, 144, 198, or 318nt in length. In further embodiments, the virus has one or more heterologous polynucleotide sequences inserted at the P-M junction and/or HN-L junction. In other embodiments, the virus has two or more heterologous polynucleotide sequences, wherein at least one heterologous polynucleotide is inserted at the P-M junction and at least one is inserted at the HN-L junction. In other embodiments, the heterologous polynucleotide sequence is a transgene encoding a polypeptide that enhances viral oncolytic properties. In yet another embodiment, the transgene encodes a cytokine, a cell surface ligand, and/or a chemokine. In other embodiments, the cytokine is selected from the group consisting of: GM-CSF, IL-2, IL-21, IL-15, IL-12, and IL-12p 70. In a specific embodiment, the cytokine is human GM-CSF. In other embodiments, the heterologous polynucleotide sequence is a transgene encoding a detectable moiety. In certain embodiments, the expression level of the detectable moiety is correlated with viral replication. In yet another embodiment, the F and HN genes of NDV are replaced by the corresponding extracellular domains of canine parainfluenza virus 5(PIV 5) or pigeon paramyxovirus type 1 (PPMV-1). In another embodiment, the virus is 73T-R116 i-hGM-CSF. In other embodiments, the attenuated virus has a mean in ovo death time (MDT) of greater than 90 hours or about 90-156 hours. In another embodiment, the attenuated virus has an intracerebral pathogenicity index of between about 0 and 0.7. In other embodimentsIn the examples, the attenuated virus has an intracerebral pathogenicity index of about 0. In yet another embodiment, the attenuated virus has a cytotoxicity of less than about 15% in HT1080 cells. In other embodiments, the attenuated virus selectively kills tumor cells with a killing efficiency of at least 10% or 15%. In another embodiment, the tumor cell killing efficiency is between about 75% and 100%.

Another aspect of the invention features, in general, a method of selectively killing a tumor cell that involves contacting the tumor cell with an attenuated newcastle disease virus described herein. In another embodiment of the invention, the tumor cell is a bladder, ovarian, brain, pancreas, prostate, sarcoma, lung, breast, cervical, liver, head and neck, stomach, kidney, melanoma, lymphoma, leukemia, thyroid, colon, and melanoma cancer cell. In yet another embodiment, the method involves administering to the subject an effective amount of an attenuated newcastle disease virus described herein. In yet another embodiment, the attenuated newcastle disease virus is delivered systemically, intraperitoneally, or intratumorally. In other embodiments, the virus is administered at about 10%⁷pfu to about 10⁹Doses of pfu were administered. In further embodiments, the virus is administered at about 10%⁹pfu to about 10¹¹Doses of pfu were administered intravenously. In other embodiments, the subject has a cancer selected from the group consisting of: bladder cancer, ovarian cancer, brain cancer, pancreatic cancer, prostate cancer, sarcoma, lung cancer, breast cancer, cervical cancer, liver cancer, head and neck cancer, gastric cancer, renal cancer, melanoma, lymphoma, leukemia, thyroid cancer, colon cancer, and melanoma.

In yet another aspect, the invention features, in general, a method of treating a neoplasia in a subject that has developed an immune response against NDV, the method involving administering to the subject an effective amount of an attenuated chimeric newcastle disease virus described herein, wherein the virus is a chimeric virus comprising the F and/or HN genes of canine parainfluenza virus 5(PIV 5) or pigeon paramyxovirus type 1 (PPMV-1), wherein the chimeric newcastle disease virus is antigenically different from NDV. In one embodiment of the invention, the method increases the level of oncolytic virus present in the subject relative to the level of oncolytic virus present in a control subject that has developed an anti-NDV epidemic response but has not received the chimeric newcastle disease virus.

Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide the skilled artisan with a general definition of a number of terms used in the present invention: singleton et al, Dictionary of microbiology and Molecular Biology (Dictionary of microbiology and Molecular Biology) (2 nd edition, 1994); cambridge scientific dictionary (The Cambridge dictionary of science and Technology) (Walker, eds., 1988); a Glossary of genetics (The Glossary of genetics), 5 th edition, R. Leehler (R.Rieger) et al (eds.), Springger Verlag (1991); and Heille (Hale) and Marham (Marham), The Harper Collins Dictionary of biology (The Harper Collins Dictionary of biology) (1991). The following terms as used herein have the following meanings, unless otherwise indicated.

By "attenuated newcastle disease virus" is meant a newcastle disease virus that selectively kills tumor cells but does not pose a threat to poultry. In one embodiment, the attenuated newcastle disease virus has an ICPI of less than about 0.4 or 0.7. In other embodiments, the attenuated newcastle disease virus has an ICPI between about 0 and 0.1.

By "heterologous polynucleotide sequence" is meant a recombinant polynucleotide that is not present in wild-type conditions.

By "detectable label" is meant a composition that, when attached to a molecule of interest, renders the latter detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, high electron density reagents, enzymes (e.g., as commonly used in enzyme-linked immunosorbent assays (ELISAs)), biotin, digoxigenin, or haptens.

By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragment thereof.

"alteration" or "change" means an increase or decrease. The alteration may be as little as 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, or 40%, 50%, 60%, or even as much as 70%, 75%, 80%, 90%, or 100%.

As used herein, the term "antibody" refers to a polypeptide or a group of polypeptides consisting of at least one binding domain formed by the folding of polypeptide chains having a three-dimensional binding space wherein the internal surface shape and charge distribution are complementary to the characteristics of an antigenic determinant of an antigen. Antibodies typically have a tetrameric form comprising two pairs of identical polypeptide chains, each pair having one "light chain" and one "heavy chain". The variable region of each light/heavy chain pair, or the variable chain polypeptide, forms the antigen binding site.

The term "mAb" refers to a monoclonal antibody. Antibodies of the invention include, but are not limited to, all-natural antibodies, bispecific antibodies; a chimeric antibody; fab, Fab', single chain V region fragment (scFv), fusion polypeptides, and non-conventional antibodies.

By "biological sample" is meant a sample obtained from a subject, including a sample of biological tissue or a fluid source obtained or collected in vivo or in situ. In particular embodiments, a biological sample includes any cell, tissue, fluid, or other biologically derived material.

By "capture reagent" is meant an agent that specifically binds to a nucleic acid molecule or polypeptide to select or isolate the nucleic acid molecule or polypeptide.

By "clinical aggressiveness" is meant the severity of the neoplasia. Aggressive neoplasias are more likely to metastasize than less aggressive neoplasias. While conservative treatment approaches are appropriate for less aggressive neoplasias, more aggressive neoplasias require more aggressive treatment regimes.

The terms "determining," "evaluating," "determining," "measuring," and "detecting," as used herein, refer to both quantitative and qualitative determinations, and as such, the term "determining" is used interchangeably herein with "determining," "measuring," and the like. Where quantitative determination is the purpose, the phrase "determining the amount of analyte and analog" is used. Where qualitative and/or quantitative determination is the purpose, the phrase "determining the level of an analyte" or "detecting" an analyte is used.

The term "subject" or "patient" refers to an animal that is the subject of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or non-human mammal, such as a non-human primate, murine, bovine, equine, canine, ovine, or feline.

The terms "decrease" or "increase" mean a negative or positive change, respectively. The change may be 5%, 10%, 25%, 30%, 50%, 75%, or even 100%.

"reference" means a standard for comparison.

"periodic" means periodic. Periodic patient monitoring includes, for example, a schedule of tests administered daily, biweekly, bimonthly, monthly, bi-yearly, or yearly.

By "severity of neoplasia" is meant the degree of pathology. The severity of neoplasia increases, for example, as the stage or grade of neoplasia increases.

Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not have 100% identity to endogenous nucleic acid sequences, but will typically exhibit substantial identity. Polynucleotides having "substantial identity" to endogenous sequences are typically capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. By "hybridizing" is meant pairing under different stringency conditions so as to form a double-stranded molecule between complementary polynucleotide sequences (e.g., genes described herein), or portions thereof. (see, for example, Wall, G.M. (Wahl, G.M.) and S.L. Berger (S.L.Berger) (1987) Methods in enzymology (Methods Enzymol.)152:399), Chimiel, A.R. (Kimmel, A.R.) (1987) Methods in enzymology (Methods Enzymol.)152: 507).

For example, a stringent salt concentration will generally be less than about 750mM sodium chloride and 75mM trisodium citrate, preferably less than about 500mM sodium chloride and 50mM trisodium citrate, and more preferably less than about 250mM sodium chloride and 25mM trisodium citrate. Low stringency hybridization can be achieved in the absence of an organic solvent (e.g., formamide), while high stringency hybridization can be achieved in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will generally include temperatures of at least about 30 ℃, more preferably at least about 37 ℃, and most preferably at least about 42 ℃. Various additional parameters, such as hybridization time, detergent (e.g., Sodium Dodecyl Sulfate (SDS)) concentration, and inclusion or exclusion of vector DNA, are well known to those of ordinary skill in the art. Different levels of stringency are achieved by combining these different conditions as required. In a preferred embodiment, hybridization will occur at 30 ℃ in 750mM sodium chloride, 75mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37 ℃ in 500mM sodium chloride, 50mM trisodium citrate, 1% SDS, 35% formamide, and 100. mu.g/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42 ℃ in 250mM sodium chloride, 25mM trisodium citrate, 1% SDS, 50% formamide, and 200.mu.g/ml ssDNA. Useful variations of these conditions will be apparent to those of ordinary skill in the art.

For most applications, the washing steps after hybridization will also differ in stringency. Washing stringency conditions can be defined by salt concentration and temperature. As mentioned above, the washing stringency can be increased by reducing the salt concentration or increasing the temperature. For example, stringent salt concentrations for the washing step will be preferably less than about 30mM sodium chloride and 3mM trisodium citrate, and most preferably less than about 15mM sodium chloride and 1.5mM trisodium citrate. Stringent temperature conditions for the washing step will generally include a temperature of at least about 25 ℃, more preferably at least about 42 ℃, and even more preferably at least about 68 ℃. In a preferred embodiment, the washing step will occur at 25 ℃ in 30mM sodium chloride, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing step will occur at 42 ℃ in 15mM sodium chloride, 1.5mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing step will occur at 68 ℃ in 15mM sodium chloride, 1.5mM trisodium citrate, and 0.1% SDS. Additional variations of these conditions will be apparent to those of ordinary skill in the art. Hybridization techniques are well known to those of ordinary skill in the art and are described, for example, in Benton (Benton) and Davis (Davis) (science 196:180, 1977); grenstein (Grunstein) and hopnens (Hogness) (proceedings of the american academy of sciences (proc. natl. acad. sci., USA)72:3961, 1975); osubel (Ausubel) et al (Molecular Biology laboratory Manual, journal of the Willi Press database, New York (Current Protocols in Molecular Biology, Wiley Interscience, New York), 2001); berger (Berger) and Kimmel (Molecular Cloning Techniques), 1987, Academic Press, New York (Academic Press, New York); and Sambrook (Sambrook) et al, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York).

By "substantially identical" is meant a polypeptide or nucleic acid molecule that exhibits at least 50% identity with respect to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). Preferably, such sequences are at least 60%, more preferably 80% or 85%, and more preferably 90%, 95%, 96%, 97%, 98%, or even 99% or more identical at the amino acid level or nucleic acid relative to the sequence used for comparison.

Typically using sequence analysis software (e.g., university of Wisconsin Biotechnology center (university of Madison 1710, Wisconsin)53705(1710University Avenue, Madison, wis.53705)) the sequence analysis software package of the genetic computing group, the BLAST, BESTFIT, GAP, or PILEUP/pretybox programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method of determining the degree of identity, the BLAST program can be used, where at e^-3And e^-100The probability scores in between indicate closely related sequences.

As used herein, "substantially pure" means that the species of interest is the predominant species present (i.e., it is more abundant than any other individual species in the composition on a molar basis), and preferably, a substantially pure fraction is the composition as follows: in which the target species comprises at least about 50% (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will include greater than about 80% of all macromolecular species present in the composition, more preferably greater than about 85%, 90%, 95%, and 99%. Most preferably, the species of interest is purified to the necessary homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) and the composition consists essentially of a single macromolecular species.

The term "treating (treating, etc.), as used herein, refers to reducing or ameliorating the disorder and/or symptoms associated therewith. It will be understood that, although not excluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated. Thus, a successful treatment may prolong patient survival or alleviate adverse symptoms.

As used herein, the terms "prevent", "preventing", "prophylactic treatment" and the like refer to reducing the likelihood of development of a disorder or condition in a subject who does not have the disorder or condition, but who is at risk of developing the disorder or condition or who is susceptible to developing the disorder or condition.

Administration refers to a single administration of the therapeutic composition. Dosage refers to the amount of therapeutically active molecule in a dose. Treatment regimen refers to the administration of a dose, schedule, and mode of one or more administrations. Cycle refers to a repeatable unit with one or more administrations in a treatment regimen. In some treatment regimens, the dose is uniform for each administration. In other treatment regimens, the dosage may not be uniform. For example, one or more loading doses may be used to increase the concentration of the therapeutic molecule in the patient to a desired level. The loading dose may be followed by one or more maintenance administrations, which generally include a lower dose (e.g., half or less of the loading dose) sufficient to maintain the desired concentration of the therapeutic molecule in the patient. One or more escalating doses may be used to escalate the concentration of the therapeutic molecule in the patient.

By "specifically binds" is meant that a compound (e.g., an antibody) recognizes and binds to one molecule (e.g., a polypeptide), but it does not substantially recognize and bind to other molecules.

Unless specifically stated or otherwise apparent from the context, the term "about" as used herein is understood to be within the normal tolerance of the art, e.g., within 2 standard deviations of the mean. About can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise apparent from the context, all numbers provided herein are approximately modified by the term.

Ranges provided herein are to be understood as shorthand for all values within the range. For example, a range of 1 to 50 should be understood to include any number, combination of numbers, or subrange from the group consisting of: 1. 2,3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

Any of the compounds, compositions, or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

As used herein, the singular forms "a", "an" and "the" include the plural forms unless the context clearly dictates otherwise. Thus, for example, reference to "a biomarker" includes reference to more than one biomarker.

The term "or," as used herein, is understood to be inclusive unless explicitly stated or otherwise evident from the context.

The term "comprising" is used herein to mean, and is used interchangeably with, the phrase "including, but not limited to".

As used herein, the terms "comprising", "including", "containing", "having" and the like have the meaning dictated by united states patent law and mean "including", "including" and the like; "consisting essentially of … (inclusive of or consensular)" has the meaning specified in U.S. patent law and the term is open-ended, allowing for the presence of more than the recited features, as long as the recited basic or new features are not changed by the presence of more than the recited, but excluding prior art embodiments.

Sequence of

An exemplary nucleotide sequence of full-length NDV virus 73T is:

tacgtataatacgactcactatagggaccaaacagagaatccgtaggttacgataaaaggcgaaggagca

attgaagttggacgggtagaaggtgtgaatctcgagtgcgagcccgaagcacaaactcgagaaagccttc

tgccaacatgtcttccgtatttgacgagtacgaacagctcctcgcgtctcagactcgccccaatggagct

catggaggaggggaaaaggggagtaccttaaaagtagacgtcccggtattcactcttaacagtgatgacc

cagaagataggtggaactttgcggtattctgcctccggattgctgttagcgaagatgccaacaaaccact

caggcaaggtgctctcatatctcttttatgctcccactcacaagtgatgaggaaccatgttgcccttgca

gggaaacagaatgaagccacattggccgtgcttgagattgatggctttgccaacggtatgccccagttca

acaataggagtggagtgtctgaagagagagcacagagattcgcgatgatagcagggtctctccctcgggc

atgcagtaatggcaccccgttcgtcacagccggggccgaagatgatgcaccagaagacatcaccgatacc

ctggagaggatcctctctatccaggcccaagtatgggtcacagtagcaaaagccatgactgcgtatgaga

ctgcagatgagtcagaaacaagacgaatcaataagtatatgcagcaaggcagggtccaaaagaaatacat

cctctaccccgtatgcaggagcacaatccaactcacgatcagacagtctcttgcagtccgcatctttttg

gttagcgagctcaagagaggccgcaacacggcaggtggtacctctacttattataacctagtaggggacg

tagactcatatatcaggaataccgggcttactgcattcttcctgacactcaagtacggaatcaacaccaa

gacatcagcccttgcacttagtagcctctcaggcgacatccagaagatgaagcagctcatgcgtttgtat

cggatgaaaggagataatgcgccgtacatgacattgcttggtgatagtgaccagatgagctttgcgcctg

ccgagtatgcacaactttactccttcgccatgggtatggcatcagtcctagataaaggtaccgggaaata

ccaatttgccagggactttatgagcacatcattctggagacttggagtagagtacgctcaggctcaggga

agtagcattaacgaggatatggctgccgagctaaagctaaccccagcagcaaggagaggcctggcagctg

ctgcccaacgagtctccgaggagaccagcagcatagacatgcctactcaacaagtcggagtcctcactgg

gctcagcgagggggggtcccaagccctacaaggcggatcgaatagatcgcaagggcaaccagaagccggg

gatggggagacccaattcctggatctgatgagagcggtagcaaatagcatgagggaagcgccaaactctg

cacagggcactccccaatcggggcctcccccaactcctgggccatcccaagataacgacaccgactgggg

gtattgattgacaaaacccagcttgcttccacaaaatcatcccaataccctcacccgtagtcgacccctc

gatttgcggccctacatgaccacaccctcaaacaaacatccccctctttcctccctccccctgctgtaca

actccgcacgccctaggtaccacaggcacaatgcggctcactaacaatcaaaacagagccgaggaaatta

gaaaaaagtacgggtagaagagggatattcagagaccagggcaagtctcccgagtctctgctctctcctc

tacctgatagattaggacaaatatggccacctttacagatgcggagatcgacgagctatttgagacaagt

ggaactgtcattgacaacataattacagcccagggtaaaccagcagagactgtgggaaggagtgcaatcc

cacatggcaaaaccaaggcgctgagcgcagcatgggagaagcatgggagcatccagccaccagccagtca

agacacccctgatcgacaggacagatctgacaaacaaccatccacacccgagcaagcgaccccgcatgac

agcccgccggccacatccgccgaccagccccccacccaggccacagacgaagccgtcgacacacagctca

ggaccggagcaagcaactctctgctgttgatgcttgacaagctcagcaataaatcatccaatgctaaaaa

gggcccatggtcgagcccccaagaggggaaccaccaacgtccgactcaacagcagggaagtcaacccagc

cgcggaaacagtcaggaaagaccacagaaccaagtcaaggccgcccctggaaaccagggcacagacgcga

acacagcatatcatggacaatgggaggagtcacaactatcagctggtgcaacccctcatgctctccgatc

aaggcagagccaagacaatacccttgtatctgcggatcatgtccagccacctgtagactttgtgcaagcg

atgatgtctatgatggaggcaatatcacagagagtaagtaaggttgactatcagctagatcttgtcttga

aacagacatcctccatccctatgatgcggtccgaaatccaacagctgaaaacatctgttgcagtcatgga

agccaatttgggaatgatgaagattctggatcccggttgtgccaacgtttcatctctgagtgatctacgg

gcagttgcccgatctcacccggttttagtttcaggccctggagacccatctccctatgtgactcaaggag

gcgaaatggcacttaataaactttcgcaaccagtgccacatccatctgaattgattaaacccgccactgc

atgcgggcctgatataggagtggaaaaggacactgtccgtgcattgatcatgtcacgcccaatgcacccg

agttcttcagccaagctcctaagcaagctagatgcagccgggtcgatcgaggaaatcaggaaaatcaagc

gccttgcactaaatggctaattaccactgccacacgtagcgggtccccgtccactcggcatcacacggaa

tctgcaccgagtccccccccgcagacctaaggtccaactctccaagtggcaatcctctctcgcttcctca

gccccactgaatgatcgcgcaaccgtaattaatctagctacattaaggattaagaaaaaatacgggtaga

attggaatgccccaattgtgccaagatggactcatctaggacaattgggctgtactttgattctgcccat

tcttctagcaacctgttagcatttccgatcgtcctacaagacacaggagatgggaagaagcaaatcgccc

cgcaatataggatccagcgccttgactcgtggactgatagtaaagaagactcagtattcatcaccaccta

tggattcatctttcaggttgggaatgaagaagccactgtcggcatgatcaatgataatcccaagcgcgag

ttactttccgctgcgatgctctgcctaggaagcgtcccaaataccggagaccttgttgagctggcaaggg

cctgtctcactatggtagtcacatgcaagaagagtgcaactaatactgagagaatggttttctcagtagt

gcaggcaccccgagtgctgcaaagctgtagggctgtggcagacaaatactcatcagcgaatgcagtcaag

cacgtgaaagcgccagagaagatccccgggagtggaaccctagaatacaaggtgaactttgtctccttga

ctgtggtaccgaagaaggatgtctacaagatcccaactgcagtattgaaggtttctggctcgagtctgta

caatcttgcgctcaatgtcactattaatgtggaggtagacccgaggagtcctttggttaaatctctgtct

aagtctgacagcggatactatgctgacctcttcttgcatattggacttatgaccaccgtagataggaagg

ggaagaaagtgacttttgacaagctagaaaagaagataaggagacttgatctatctgtcgggctcagtga

tgtgctcggaccttccgtgctggtaaaagcaagaggtgcacggaccaagcttttggcacctttcttctct

agcagtgggacagcctgctatcccatagcaaatgcctctccccaggtggccaagatactctggagtcaaa

ccgcgtgcctgcggagcgttaaaatcattatccaagcaggtacccaacgcgctgtcgcagtgaccgctga

ccacgaggttacctctactaagctggagaaggggcacacccttgccaaatacaatccttttaagaaataa

gctgcgtttctgagattgcgctccgcccactcacccagagcatcatgacaccaaaaactaatctgtcttg

attatttacagttagtttacctgtctatcaaattagaaaaaacacgggtagaagattctggatcccggtt

ggcgccttctaggtgcaagatgggccccagaccttctaccaagaacccagcacctatgatgctgactgtc

cgggtcgcgctggtactgagttgcatctgtccggcaaactccattgatggcaggcctcttgcggctgcag

gaattgtggtaacaggagacaaagcagtcaacatatacacctcatcccagacaggatcaatcatagttaa

gctcctcccaaacctgcccaaggataaggaggcatgtgcgaaagcccccttggatgcatacaacaggaca

ttgaccactttgctcaccccccttggtgactctatccgtaggatacaagagtctgtaactacatctggag

ggaggagacagaaacgctttataggcgccattattggcggtgtggctcttggagttgcaactgctgcaca

aataacagcggccgcagctctgatacaagccaaacaaaatgctgccaacatcctccgacttaaagagagc

attgccgcaaccaatgaggccgtgcatgaggtcactgacggattatcgcaactagcagtggcagttggga

agatgcagcagtttgtcaatgaccaatttaataaaacaactcaggaattaggctgcatcagaattgcaca

gcaagttggcgtagagctcaacctgtatctaaccgaattgactacagtattcggaccacaaatcacttca

cctgccttaaacaagctgactattcaggcactttacaatctagctggtgggaatatggatcacttgttga

ctaagttaggtgtagggaacaatcaactcagctcattaatcggtagcggcttaatcaccggcaaccctat

tctgtacgactcacagactcaactcttgggtatacaggtaactctaccttcagtcgggaacctaaataat

atgcgtgccacctacttggaaaccttatccgtaagcacaaccaggggatttgcctcggcacttgtcccaa

aagtggtgacacaggtcggttctgtgatagaagaacttgacacctcatattgtatagaaaccgacttgga

tttatattgtacaagaatagtaacattccctatgtcccctggtatttattcctgcttgagcggcaataca

tcggcctgtatgtactcaaagaccgaaggcgcactcactacgccatacatgactatcaaaggctcagtca

tcgctaactgcaagatgacaacatgtagatgtgtaaaccccccgggtatcatatcgcaaaactatgggga

agccgtgtctctaatagataagcaatcatgcaatgttttatccttagacgggataactttaaggctcagt

ggggaattcgatgcaacttatcagaagaatatctcaatacaagattctcaagtaataataacaggcaatc

ttgatatctcaactgagcttgggaatgtcaacaactcgatcagtaatgctttgaataagttagaggaaag

caacagcaaactagacaaagtcaatgtcaaactgaccagcacatctgctctcattacctatatcgttttg

actatcatatctcttgtttttggtatacttagcctggttctagcatgctacctaatgtacaagcaaaagg

cgcaacaaaagaccttattatggcttgggaataataccctagatcagatgagagccactacaaaaatgtg

aacacagatgaggaacgaaggtatccctaatagtaatttgtgtgaaagttctggtagtctgtcagttcgg

agagtttagaaaaaactaccggttgtagatgaccaaaggacgatatacgggtagaacggtaagagaggcc

gcccctcaattgcgagccgggcttcacaacctccgttctaccgcttcaccgacagcagtcctcagtcatg

gaccgcgcagttagccaagttgcgttagagaatgatgaaagagaggcaaaaaatacatggcgcttgatat

tccggattgcaatcttactcttaacagtagtgaccttagctacatctgtagcctcccttgtatatagcat

gggggctagcacacctagcgaccttgtaggcataccgaccaggatttccagggcagaagaaaaaattaca

tctgcacttggttccaatcaagatgtagtagataggatatataagcaagtggcccttgagtctccgttgg

cattgttaaacactgagatcacaattatgaacgcaataacatctctctcttatcagattaatggagctgc

gaacaacagcgggtggggggcacctatccatgacccagattttatcggggggataggcaaagaactcatt

gtagatgatgctagtgatgtcacatcattctatccctctgcatttcaagaacatctgaattttatcccgg

cgcctactacaggatcaggttgcactcggttaccttcatttgacatgagtgctacccattactgctacac

tcataatgtaatattgtctggatgcagagatcactcacactcacatcagtatttagcacttggtgtgctc

cggacatctgcaacagggaggatattcttttctactctgcgttccatcaatctggatgacacccaaaatc

ggaagtcttgcagtgtgagtgcaactcccttaggttgtgatatgctgtgctcgaaagtcacggagacaga

ggaagaagattataactcagctgtccctacgctgatggtacatgggaggttagggttcgacggccaatac

cacgaaaaggacctagacgtcacaacattatttgaggactgggtggccaactacccaggagtagggggtg

gatcttttattgacagccgcgtatggttctcagtctacggagggctgaaacccaactcacccagtgacac

tgtacaggaagagaaatatgtaatatacaagcgatacaatgacacatgcccagatgagcaagactaccag

atccgaatggccaagtcttcgtataagcccgggcggtttggtgggaaacgcatacagcaggctatcttat

ctatcaaggtgtcaacatctttgggcgaagacccagtactgactgtaccgcccaacacagtcacactcat

gggggccgaaggcagaattctcacagtagggacatctcatttcttgtatcagcgagggtcatcatacttc

tctcccgcgttattatatcctatgacagtcagcaacaaaacagccactcttcatagtccctatacattca

atgccttcactcggccaggtagtatcccttgccaggcttcagcaagatgccccaactcgtgtgttactgg

agtctatacagatccatatcccctaatcttctataggaaccacaccttgcgaggggtattcgggacaatg

cttgatggtgtacaagcaagactcaatcctgcgtctgcagtattcgacagcacatcccgcagtcgcacaa

cccgagtgagttcaagcagcaccaaagcagcatacacaacatcaacctgttttaaagttgtcaagaccaa

taagacctattgtctcagcattgctgaaatatctaatactctctttggagaattcagaatcgtcccgtta

ctagttgagatcctcaaaaatgatggggttagagaagccaggtctggttagttgagtcaactatgaaaga

gctggaaagatggcattgtatcacctatcttccgcgacaccaagaatcaaactgaatgccggtgtgagct

cgaattccatgtcgccagttgactacaatcagccagtgctcatgcgatcagatcaagtcttgtcaatagt

ccctcgattaagaaaaaatgtaagtggcaatgagatacaaggcaaaacagctcatggtaaatagtacggg

taggacatggcgagctctggtcctgaaagggcagagcatcagattatcctaccagagtcacacctgtctt

caccattggtcaagcacaaactactttattactggaaattaactgggttaccgcttcctgatgaatgtga

cttcgaccacctcattctcagcagacaatggaaaaaaatacttgaatcggcctctcctgatactgagaga

atgataaaactcggaagggcagtacaccaaactctcaaccacaattctagaataaccggagtactccacc

ccaggtgtttagaagaactggctagtattgaggtccctgattcaaccaacaaatttcggaagattgagaa

gaagatccaaattcacaacacgagatatggagaaatgttcacaaggctgtgtacgcatatagagaagaaa

ctgctggggtcatcctggtctaacaatgtcccccggtcagaggagttcaacagcatccgtacggatccgg

cattctggtttcactcaaaatggtccacagccaagtttgcatggctccatataaaacagatccagaggca

tctgattgtggcagctaggacaagggctgcggccaacaaattggtgatgctaacccataaggtaggccaa

gtctttgtcactcctgaacttgtcattgtgacgcatacgaatgagaacaagttcacatgtcttacccagg

aacttgtattgatgtatgcagatatgatggagggcagagatatggtcaacataatatcaaccacggcggt

gcatctcagaagcttatcagagaaaattgatgacattttgcagttaatagacgctctggcaaaagacttg

ggtaatcaagtctacgatgttgtatcactaatggagggatttgcatacggagctgtccagctgctcgagc

cgtcaggtacatttgcaggagatttcttcgcattcaacctgcaggagcttaaagacattctaatcggcct

cctccccaatgatatagcagaatccgtgactcatgcaatagctactgtattctctggtttagaacagaat

caagcagctgagatgttgtgcctgttgcgtctgtggggtcacccactgcttgagtcccgtattgcagcaa

aggcagtcaggagccaaacgtgcgcaccgaaaatggtggactttgatatgatccttcaggtactgtcttt

cttcaagggaacaatcatcaacggatacagaaagaagaatgcaggtgtgtggccgcgagtcaaagtggat

acaatatatgggaaggtcattgggcaactacatgcagattcagcagagatttcacacgatatcatgttga

gagagtataagagtttatctgcacttgaatttgagccatgtatagaatacgaccctgtcactaacctgag

catgttcctaaaagacaaggcaatcgcacaccctaacgataattggcttgcctcgtttaggcggaacctt

ctctccgaagaccagaagaaacatgtaaaagaagcaacttcgactaatcgcctcttgatagagtttttag

agtcaaatgattttgatccatataaagagatggaatatctgacgaccctggagtaccttagagatgacga

tgtggcagtatcatactcgctcaaagagaaggaagtgaaagttaatggacggatcttcgctaagctgaca

aagaagttaaggaactgtcaggtgatggcggaagggatcctagccgaccagattgcacctttctttcagg

gaaatggagtcattcaggatagcatatctttgaccaagagtatgctagcgatgagtcaactgtcttttaa

cagcaataagaaacgtatcactgactgtaaagaaagagtatcttcaaaccgcaatcatgatccgaagagc

aagaaccgtcggagagttgcaaccttcataacgactgacctgcaaaagtactgtcttaattggagatatc

agacaatcaaactgttcgctcatgccatcaaccagttgatgggcctacctcacttcttcgagtggattca

cctaagactgatggacactacaatgttcgtaggagaccctttcaatcctccaagtgaccctactgactgt

gacctctcaagagtccctaatgatgacatatatattgtcagtgccagagggggtatcgaaggattatgtc

agaagctatggacaatgatctctattgctgcaatccaacttgctgcagctagatcgcattgtcgcgttgc

ctgtatggtacagggtgataatcaagtaatagcagtaacgagagaggtaagatcagacgactctccggag

atggtgttgacacagttgcatcaagccagtgataatttcttcaaggaattaattcatgtcaatcatttga

ttggccataatttgaaggatcgtgaaaccatcaggtcagacacattcttcatatacagcaaacgaatctt

caaagatggagcaatcctcagtcaagtcctcaaaaattcatctaaattagtactagtatcaggtgatctc

agtgaaaacaccgtaatgtcctgtgccaacattgcctctactgtagcacggctatgcgagaacgggcttc

ccaaggacttctgttactatttaaactatataatgagttgcgtgcagacatactttgactctgagttctc

catcaccaacaattcgcaccccgatcttaaccagtcgtggattgaggacatctcttttgtgcactcatat

gttctgactcctgcccaattagggggacttagtaaccttcaatactcaaggctctacactagaaatatcg

gtgacccggggactactgcttttgcagagatcaagcgactagaagcagtgggattactgagtcctaacat

tatgactaatatcttaactaggccgcctgggaatggagattgggccagtctttgcaacgacccatactct

ttcaattttgagactgttgcaagcccaaacattgttcttaagaaacatacgcaaagagtcctatttgaaa

cttgttcaaatcccttattgtctggagtgcacacagaggataatgaggcagaagagaaggcattggctga

attcttgcttaatcaagaggtgattcatccccgcgttgcgcatgctatcatggaggcaagctctgtaggt

aggagaaagcaaattcaagggcttgttgacacaacaaacaccgtaattaagattgcacttactaggaggc

cactaggcatcaagaggctgatgcggatagtcaattattctagcatgcatgcaatgctgtttagagacga

tgttttttcctccaatcgatccaaccaccccttagtctcttctaatatgtgttctctgacactggcagac

tatgcacggaatagaagctggtcacctttgacgggaggcaggaaaatactgggtgtatctaatcctgata

cgatagaactcgtagagggtgagattcttagtgtaagcggagggtgcacaagatgtgacagcggagatga

acagtttacttggttccatcttccaagcaatatagaattgaccgatgacaccagcaagaatcctccgatg

agagtaccatatctcgggtcaaagacacaggagaggagagctgcctcacttgcgaaaatagctcatatgt

cgccacatgtgaaggctgccctaagggcatcatccgtgttgatctgggcttatggggataatgaagtaaa

ttggactgctgctcttacgattgcaaaatctcggtgtaatataaacttagagtatcttcggttattgtcc

cctttacccacggctgggaatcttcaacatagactagatgacggtataactcagatgacattcacccctg

catctctctacagggtgtcaccttacattcacatatccaatgattctcaaaggctattcactgaagaagg

agtcaaagaggggaatgtggtttatcaacagatcatgctcttgggtttatctctaatcgaatcgatcttt

ccaatgatgacaaccaggacatatgatgagatcacattgcatctacatagtaaatttagttgctgtatca

gggaagcacctgttgcggttcctttcgagctacttggggtggcaccggagctaaggacagtgacctcaaa

taagtttatgtatgatcctagccctgtatcggagggagactttgcgagacttgacttagctatcttcaag

agttatgagcttaatctggagtcatatcccacgatagagctaatgaacattctttcaatatccagcggga

agttgattggccagtctgtggtttcttatgatgaagatacctccataaagaatgacgccataatagtgta

tgacaatacccgaaattggatcagtgaagctcagaattcagatgtggtccgcttatttgaatatgcagca

cttgaagtgctcctcgactgttcttaccaactctattatctgagagtaagaggcctagacaatattgtct

tatatatgggtgatttatacaagaatatgccaggaattctactttccaacattgcagccacaatatctca

tcccgtcattcattcaaggttacatgcagtgggcctggtcaaccatgacggatcacaccaacttgcagat

acggattttatcgaaatgtctgcaaaactgttagtatcttgcactcgacgtgtgatctccggcttatatt

cagggaataagtatgatctgctgttcccatctgtcttagatgataacctgaatgagaagatgcttcagct

gatatcccggttatgctgtctgtacacggtactctttgctacaacaagagaaatcccgaaaataagaggc

ttatctgcagaagagaaatgttcagtacttactgagtatctactgtcggatgctgtgaaaccattactta

gccctgatcaggtgagctctatcatgtctcctaacataattacattcccagctaatctgtactacatgtc

tcggaagagcctcaatttgatcagggaaagggaggacagggatactatcctggcgttgttgttcccccaa

gagccattattagagttcccttctgtgcaagatattggtgctcgagtgaaagatccattcacccgacaac

ctgcggcatttttgcaagagttagatttgagtgctccagcaaggtatgacgcattcacacttagtcagat

tcatcctgagctcacatcaccaaatccggaggaagactacttagtacgatacttgttcagaggaataggg

gctgcatcctcctcttggtataaggcatcccatctcctttctgtacccgaggtaagatgtgcaagacacg

ggaactccttatacttagctgaaggaagcggagccatcatgagtcttctcgaactgcatataccacatga

aactatctattacaatacgctcttttcaaatgagatgaaccccccgcagcgacatttcgggccgacccca

acccagtttttgaattcggttgtttataggaacctacaggcggaggtaacatgcaaggatggatttgtcc

aagagttccgtccactatggagagaaaatacagaggaaagcgacctgacctcagataaagcagtggggta

tattacatctgcagtgccctacagatctgtatcattgctgcattgtgacattgaaatccctccagggtcc

aatcaaagcttactagatcaactagctatcaatttatctctgattgccatgcattccttaagggagggcg

gggtagtgatcatcaaagtgttgtatgcaatgggatactactttcatctactcatgaacttgttcgctcc

gtgttccacaaaaggatacattctctctaatggttatgcatgtagaggggatatggagtgttacctggta

tttgtcatgggttacctgggcgggcctacatttgtacacgaggtggtgaggatggcaaaaactctggtgc

agcggcacggtacgcttttgtccaaatcagatgagatcacactgaccaggttattcacctcacagcggca

gcgtgtgacagacatcctatccagtcctttaccaagattaataaagtacttgagaaagaatattgacact

gcgctgattgaagctgggggacagcccgtccgtccattctgtgcagagagtttggtgagcacgctagcgg

acataactcagataacccagatcattgctagtcacattgacacagtcatccggtctgtgatatatatgga

agctgagggtgatctcgctgacacagtttttctatttaccccttacaatctctctactgacgggaaaaag

agaacatcacttaaacagtgcacgagacagatcctagaggttacaatactgggtcttagagtcgaagatc

tcaataaaataggcgatgtaatcagcctagtgcttaaaggcatgatctccatggaggaccttatcccact

aaggacatacttgaagcatagtacctgccctaaatatttgaaggctgtcctaggtattaccaaacttaaa

gaaatgtttacagacacctctgtattgtacttgactcgtgctcaacaaaaattctacatgaaaactatag

gcaatgcagtcaaaggatattacagtaactgtgactcttaacgaaaatcacatattaataggctcttttt

ctggccaattgtatccttggtgatttaattatactatgttagaaaaaagttgaactctgactccttagag

ctcgaattcgaactcaaataaatgtcttaaaaaaaggttgcgcacaatttttcttgagtgtagtcttgtc

attcaccaaatctttgtttggtggccggcatggtcccagcctcctcgctggcgccggctgggcaacattc

cgaggggaccgtcccctcggtaatggcgaatgggacgtcgacagctaacaaagcccgaaggaagtgagtt

gctgctgccaccgttgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttt

tgctgaaaggagtcgtggagacgttgtttaaac

an exemplary nucleotide sequence of the wt F protein is as follows, wherein the underlined sequence indicates the nucleotide sequence of the F protein cleavage site:

atgggccccagaccttctaccaagaacccagcacctatgatgctgactgtccgggtcgcgctggtactgagttgcatctgtccggcaaactccattgatggcaggcctcttgcggctgcaggaattgtggtaacaggagacaaagcagtcaacatatacacctcatcccagacaggatcaatcatagttaagctcctcccaaacctgcccaaggataaggaggcatgtgcgaaagcccccttggatgcatacaacaggacattgaccactttgctcaccccccttggtgactctatccgtaggatacaagagtctgtaactacatctggagggaggagacagaaacgctttataggcgccattattggcggtgtggctcttggagttgcaactgctgcacaaataacagcggccgcagctctgatacaagccaaacaaaatgctgccaacatcctccgacttaaagagagcattgccgcaaccaatgaggccgtgcatgaggtcactgacggattatcgcaactagcagtggcagttgggaagatgcagcagtttgtcaatgaccaatttaataaaacaactcaggaattaggctgcatcagaattgcacagcaagttggcgtagagctcaacctgtatctaaccgaattgactacagtattcggaccacaaatcacttcacctgccttaaacaagctgactattcaggcactttacaatctagctggtgggaatatggatcacttgttgactaagttaggtgtagggaacaatcaactcagctcattaatcggtagcggcttaatcaccggcaaccctattctgtacgactcacagactcaactcttgggtatacaggtaactctaccttcagtcgggaacctaaataatatgcgtgccacctacttggaaaccttatccgtaagcacaaccaggggatttgcctcggcacttgtcccaaaagtggtgacacaggtcggttctgtgatagaagaacttgacacctcatattgtatagaaaccgacttggatttatattgtacaagaatagtaacattccctatgtcccctggtatttattcctgcttgagcggcaatacatcggcctgtatgtactcaaagaccgaaggcgcactcactacgccatacatgactatcaaaggctcagtcatcgctaactgcaagatgacaacatgtagatgtgtaaaccccccgggtatcatatcgcaaaactatggggaagccgtgtctctaatagataagcaatcatgcaatgttttatccttagacgggataactttaaggctcagtggggaattcgatgcaacttatcagaagaatatctcaatacaagattctcaagtaataataacaggcaatcttgatatctcaactgagcttgggaatgtcaacaactcgatcagtaatgctttgaataagttagaggaaagcaacagcaaactagacaaagtcaatgtcaaactgaccagcacatctgctctcattacctatatcgttttgactatcatatctcttgtttttggtatacttagcctggttctagcatgctacctaatgtacaagcaaaaggcgcaacaaaagaccttattatggcttgggaataataccctagatcagatgagagccactacaaaaatgtga

exemplary amino acid sequence of wild-type F protein, wherein underlined sequence indicates the amino acid sequence of the F protein cleavage site:

mgprpstknpapmmltvrvalvlscicpansidgrplaaagivvtgdkavniytssqtgsiivkllpnlpkdkeacakapldaynrtlttlltplgdsirriqesvttsggrrqkrfigaiiggvalgvataaqitaaaaliqakqnaanilrlkesiaatncavhevtdglsqlavavgkmqqfvndqfnkttqelgciriaqqvgvelnlyltelttvfgpqitspalnkltiqalynlaggnmdhlltklgvgnnqlssligsglitgnpilydsqtqllgiqvtlpsvgnlnnmratyletlsvsttrgfasalvpkvvtqvgsvieeldtsycietdldlyctrivtfpmspgiysclsgntsacmyaktegalttpymtikgsvianckmttcrcvnppgiisqnygeavslidkqscnvlsldgitlrlsgefdatyqknisiqdsqviitgnldistelgnvnnsisnalnkleesnskldkvnvkltstsalityivltiislvfgilslvlacylmykqkaqqktllwlgnntldqmrattkm

exemplary nucleotide sequences for mouse GM-CSF are as follows:

Atgtggctgcagaacctgctgttcctgggcatcgtggtgtacagcctgagcgcccctaccagatcccccatcaccgtgaccagaccctggaaacatgtggaagccatcaaagaggccctgaatctgctggacgacatgcccgtgaccctgaacgaagaggtggaagtggtgtccaacgagttcagcttcaagaaactgacctgcgtgcagacccggctgaagatctttgagcagggcctgagaggcaacttcaccaagctgaagggcgctctgaacatgaccgccagctactaccagacctactgcccccccacccccgagacagattgcgagacacaagtgaccacctacgccgacttcatcgacagcctgaaaaccttcctgaccgacatccccttcgagtgcaagaaacccggccagaagtga

an exemplary amino acid sequence of mouse GM-CSF is as follows:

mwlqnllflgivvyslsaptrspitvtrpwkhveaikcalnllddinpvtlneevevvsnefsfkkltcvqtrlkiteqglrgnftklkgalnmtasyyqtycpptpeetdcetqvttyadfidslktfltdipfeckkpgqk

exemplary nucleotide sequences for human GM-CSF are as follows:

Atgtggctgcagagcctgctgctgctgggcacagtggcctgtagcatctctgcccctgccagaagccctagccctagcacacagccctgggagcatgtgaacgccatccaggaagccagacggctgctgaacctgagcagagacacagccgccgagatgaacgagacagtggaagtgatctccgagatgttcgatctgcaagagcctacctgcctgcagacccggctggaactgtacaagcagggcctgagaggcagcctgaccaagctgaagggacccctgaccatgatggccagccactacaagcagcactgcccccccacacccgagacaagctgtgccacccagatcatcaccttcgagagcttcaaagagaacctgaaggacttcctgctcgtgatccccttcgactgctgggagcccgtgcaggaatga

an exemplary amino acid sequence of human GM-CSF is as follows:

mwlqsllllgtvacsisaparspspstqpwehvnaiqearrllnlsrdtaaemnetvevisemfdlqeptclqtrlelykqglrgsltklkgpltmmashykqhcpptpetscatqiitfesfkenlkdfllvipfdcwepvqe

brief description of the drawings

FIG. 1 depicts the construction of NDV73T antigenomic cDNA virus strain 73T. The NDV sequences in GenBank were aligned to obtain a consensus sequence to design DNA oligonucleotides for RT-PCR of viral RNA. Six subgenomic cDNA fragments generated by high fidelity RT-PCR were assembled into the pUC19 vector. The full-length cDNA of NDV73T was designated p 73T. The nucleotide and deduced amino acid sequence of the F Protein Cleavage Site (FPCS) in 73T were modified to those of NDV lassota virus strain (lento) and gB of Cytomegalovirus (CMV) (S116). The double slash indicates the cleavage site of the F protein. In addition, the 73T strain cDNA plasmid (p73T) contained a 27 nucleotide (nt) T7RNA polymerase promoter at the 5 'end, 189 nt containing the HDV antigenomic ribozyme sequence and a T7RNA polymerase transcription termination signal at the 3' end. To generate non-virulent NDV, the sequence encoding the protease cleavage site of the fusion protein is modified by site-directed mutagenesis to those of the (lentogenic) or cytomegalovirus glycoprotein b (gb) of the non-virulent NDV LaSota strain (S116).

Fig. 2A and 2B depict the insertion of one or more transgene cassettes into the NDV73T genome. FIG. 2A shows the insertion of a transgene at the P-M junction. An AfeI restriction site was introduced at nt3148 in the subcloning plasmid containing the SacII-PmlI fragment. cDNAs encoding codon-optimized human or mouse granulocyte-macrophage colony stimulating factor (GM-CSF) or interleukin 2 (IL-2). The inserted gene cassette contains the gene end (GE; 5'-TTAAGAAAAAA-3'), the intergenic nucleotide (T), the gene promoter sequence (GS; 5'-ACGGGTAGA-3') and the Open Reading Frame (ORF) of the transgene. In addition, ten nucleotides (5'-cgccgccacc-3') were inserted upstream of the start site to introduce the Kozak sequence. The SacII-PmlI fragment from the resulting plasmid was transferred into the plasmid r73T and designated P73T-P1. Furthermore, FIG. 2A shows the insertion of the transgene at the HN-L junction between the HN ORF and the gene end signal (GE) sequence of HN, introducing the AfeI restriction site at nt 8231 in the plasmid containing the AgeI-XbaI fragment. The gene cassette was generated by PCR using a pair of phosphate sense and antisense primers (table 3) and inserted into the AfeI site. The AgI-XbaI fragment from the resulting plasmid was transferred to plasmid p73T, yielding p73T-HN 1. The Full Length (FL)73TcDNA comprising the transgene at the P-M or HN-L junction was designated P73T-P1 or P73T-HN1, respectively. FIG. 2B shows the insertion of two transcription cassettes into the P-M junction. An AfeI site was introduced at the end of the ORF of GM-CSF (nt 3619). The IL-2ORF was amplified using a pair of phosphate sense and antisense primers containing GE and GS sequences and inserted at the AfeI site. The SacII-PmlI fragment from the resulting plasmid, including the GM-CSF and IL-2 transcription cassettes, was replaced back into plasmid r73T, yielding P73T-P2.

FIGS. 3A-3C show recovery of infectious recombinant NDV strain 73T (r73T) with modified FPCS and depict in vitro F protein cleavage and fusion activity. FIG. 3A shows how NDV73T NP, P, L and antigen cDNA (p73T-lento or p73T-S116) can be cloned under the control of T7RNA polymerase promoter and terminator. The four plasmids were co-transfected into RNA polymerase expressing cell lines. The recovered virus was designated r73T-lento or r 73T-S116. R73T-lento and r73T-S116 were passaged in Vero cells (Vero cells) with medium with or without trypsin supplementation. Growth of r73T-lento is trypsin dependent, however r73T-S116 can be grown in medium without trypsin supplementation. To assess the genetic stability of FPCS in r73T-S116 and transgenes, r73T-S116 with or without hGM-CSF at the P-M junction was further passaged at MOI0.01 in Vero and human fibrosarcoma HT1080 cells for 10 generations in medium without trypsin supplementation. Mutations (R113K and/or Q114M) occurred at 7 th generation in FPCS, and the S116R mutation was detected at 9 th generation. At passage 10, F, HN and the transgene were sequenced, and no additional mutations were presentAnd (4) becoming discovered. FIGS. 3B and 3C show the effect of F Protein Cleavage Site (FPCS) mutations on cell fusion and F protein cleavage in vitro. For the construction of plasmids co-expressing the two transgenes, GFP and NDVF or HN genes, the protein open reading frame of the NDVF or HN gene was amplified by PCR and cloned into the plasmid pvtro 2-neo-MCS (Invitrogen) under the control of the Cytomegalovirus (CMV) promoter. The next day, 293T cells were plated at 5X10⁵Cells/well were seeded in 6-well plates for transfection. FIG. 3B depicts cells transfected with 2. mu.g of NDVF plasmid DNA for one day and obtained in lysis buffer with protein for Western blot analysis (using anti-NDV F-specific polyclonal antiserum). NDVF protein with a weakly toxic cleavage site and S116 was not cleaved, only F0 was detected. The F proteins of R116 and S116-KM were partially cleaved as indicated by the appearance of the F1 protein band. FIG. 3C shows cells that were co-transfected with different F plasmids along with the wtHn plasmid and that were tested for fusion formation by fluorescence microscopy. The wt F protein is most effective in fusion formation.

FIG. 4 is a table summarizing the characteristics of r73T-lento and r73T-S116 derivatives.^aAll viruses contained hGM-CSF at the P-M junction.^bAmino acids in FPCS that differ from FPCS-S116 are underlined.^cPlaque formation in veronol cells without trypsin in the overlay after 36 hours incubation and visualized at 10-fold magnification.^dMean time to death (MDT) in eggs.^ePathogenicity of NDV in pathogen-free chickens at 1 day of age, expressed as the intracerebral pathogenicity index (ICPI). ICPI measurements were performed at the National Veterinary Services Laboratories (NVSL) (emms, iowa).^fCytotoxic effect of the virus on human fibrosarcoma HT1080 cells after infection with a multiplicity of infection (MOI) of 0.01 72 hours post-infection. The relative percentage of viable cells was determined by comparing each sample to untreated cells considered to be 100% viable. The data presented in the table are relative percentages of dead cells.^gInfection at an MOI of 0.01Followed by 3-5 days of virus grown in Vero cells and cultured in OPTI-MEM without trypsin supplementation at 37 ℃.^hViruses that grow in chicken embryo eggs. 10-11 day old chick embryos were infected with 1,000pfu of r 73T. Amniotic fluid was obtained after incubation at 37 ℃ for 72 hours. Infectious virus titers in veronol cells were determined by plaque assay.

FIGS. 5A and 5B depict strategies for attenuating the virulence of the R73T-R116 virus in chickens. FIG. 5A depicts the insertion of a transgene at P-M junction (1) and HN-L junction (2), and the elongation of the HN-L intergenic region by insertion of a non-coding sequence (3). Insertion of the transgene cassette at the P-M junction is the same as shown in FIG. 2A. The second transgene cassette comprises the L gene promoter sequence (GS; 5'-ACGGGTAGA-3'), the Open Reading Frame (ORF) of the transgene, sequences from the 3 'untranslated region of the L gene (italics) and the L gene end sequence (GE; 5' -TTAAGAAAAAA-3). The non-coding sequences for extending the HN-L junction are taken from paramyxovirus type 1 (APMV-1), Respiratory Syncytial Virus (RSV) or random sequences with no sequence identity or homology to known sequences. The insertion sequence may be in the range of 60-318 nt. Insertion of a second transgene at HN-L allows the virus to express both transgenes (e.g., hGM-CS and GFP). FIG. 5B depicts the sequence inserted at the HN-L junction.

FIG. 6A is a table summarizing the characteristics of R73T-R116 derivatives.^aAll viruses contained hGM-CSF at the P-M junction.^bThe sequence inserted at the HN-L junction is shown in FIG. 5B.^cPlaque formation in veronol cells without trypsin in the overlay after 36 hours incubation and visualized at 10-fold magnification.^dMean time to death (MDT) in eggs.^ePathogenicity of NDV in pathogen-free chickens at 1 day of age, expressed as the intracerebral pathogenicity index (ICPI). ICPI measurements were performed at the National Veterinary Services Laboratory (NVSL) (emms, iowa).^fCytotoxic effect of the virus on human fibrosarcoma HT1080 cells after infection with a multiplicity of infection (MOI) of 0.01 72 hours post-infection. The relative percentage of viable cells was determined by comparing each sample to untreated cells considered to be 100% viable. The data present in the table areRelative percentage of dead cells.^gViruses grown in Vero cells after infection at an MOI of 0.01 and cultured in OPTI-MEM without trypsin supplementation at 37 ℃ for 3-5 days.^hViruses that grow in chicken embryo eggs. 10-11 day old chick embryos were infected with 1000pfu of r 73T. Amniotic fluid was obtained after incubation at 37 ℃ for 72 hours. Infectious titer in veronol cells was determined by plaque assay.

FIGS. 6B-6E are graphs showing the growth kinetics of recombinant NDV in DF-1 and Verilor cells. DF-1 and vilo cells in six-well plates were infected with each indicated virus at a multiplicity of infection (m.o.i.) of 5.0 (single cycle, fig. 6B and 6C) or 0.001 (multiple cycle, fig. 6C and 6D). Infected cell culture supernatants were collected every 10 hours until 50 hours post infection and virus titers were determined by plaque assay.

FIGS. 6F and 6G show viral RNA and protein synthesis in DF-1 cells. DF-1 cells were infected with each indicated virus at 5.0 m.o.i., incubated for 20 hours, total intracellular RNA was extracted for northern blot analysis (fig. 6F), and a second set of infected cells was examined by western blot for protein synthesis (fig. 6G). RNA was separated by electrophoresis in formaldehyde agarose gel, transferred to nitrocellulose membrane, and hybridized with biotin-labeled RNA probes specific for HN, NP, P, and L genes. Positive RNA probes were used to detect viral genomic RNA in the L gene. Total proteins were separated on SDS-PAGE and blotted with anti-NP, F, HN and L sera. Total protein loaded on the gel was detected by actin-specific antibodies. Recombinant NDV R116i with a 198nt insertion between HN and L intergenic sequences had overall reduced RNA and protein synthesis in DF-1 cells.

FIGS. 6H and 6I depict viral RNA and protein synthesis in Vero cells. FIG. 6H shows northern blot analysis of RNA synthesis. Vilo cells were infected with virus at 5.0 m.o.i., and incubated for 20h to extract total intracellular RNA. RNA was separated by electrophoresis in a formaldehyde agarose gel, transferred to nitrocellulose membrane, and genomic RNA was detected by hybridization with biotin-labeled RNA probes specific for the NP and L genes and the sense L gene RNA. FIG. 6I depicts a Western blot analysis of viral protein synthesis in infected Vero cells. Total proteins were separated on SDS-PAGE, transferred to nitrocellulose membrane, and blotted with anti-NP, F, HN, and L sera. Total protein loaded on the gel was detected by actin-specific antibodies. The R116i virus with the 198nt insertion had reduced genomic RNA compared to S116 and wt NDV, but greatly increased NP mRNA. The L mRNA level was too low to be distinguished. In vilo cells, proteins upstream of the L gene are upregulated, but L protein levels are decreased.

FIGS. 6J and 6K show a comparison of protein synthesis by Western blot analysis in DF-1 and Verilor cells infected at low multiplicity of infection. DF-1 and vilo cells were infected with each virus indicated at 0.001 m.o.i, incubated for 72h and obtained in protein lysis buffer. Total proteins were separated on SDS-PAGE, transferred to nitrocellulose membrane, and blotted with anti-NP, F, HN, and L sera. The protein loaded on the gel was detected by an antibody against actin. L protein levels were reduced in both cell lines, however, proteins upstream of the L gene were down-regulated in DF-1 cells but up-regulated in vilo cells.

FIG. 6L shows viral protein synthesis in infected human cells compared to F-1 cells. Human HT1080, hela cells and DF-1 cells were infected with each virus indicated at m.o.i. of 5.0, incubated for 20h and obtained in proteolytic lysis buffer. Proteins were separated on SDS-PAGE, transferred to nitrocellulose membrane, and blotted with anti-HN and anti-L sera. HN protein expression of R116i with a 198nt insertion between HN and L junctions was increased in HT1080 and hela cells, but decreased in DF-1 cells. L protein was reduced in all three cell lines infected with R116i-198 or 198 RSV.

FIG. 7 is a graph depicting the growth kinetics of the r73T virus in chicken embryos. To determine the growth conditions for the r73T virus, growth kinetics studies were performed in ovo. Embryonated chicken eggs were infected with the indicated virus at 100, 1000, 10,000 or 100,000 PFU/egg and incubated for 2,3 or 4 days (73T wt, top left; R116, top right; R116i-318, middle left; R116i-198-RSV, middle right; R116 i-198-random, bottom left; S116-KM, bottom right). Allantoic fluid was obtained and virus titer was determined by FFA. Inoculation with 100 FFU/egg had low titers at day 1, but peak titers were reached at day 2. In summary, most viruses reached a peak titer of about 8logs/mL on day 2, regardless of the dose used for vaccination. R116i-198 had the lowest titer, with low inoculum size yielding the highest yield on day 2, indicating the possible accumulation of defective particles. The R116 i-318-APMV-insertion also had a lower peak titer of about 7 logs. R116i-198-RSV reached a peak titer of 7.5 logs. S116, re-derived from reverse genetics, did not reduce virus production in eggs. Thus, among the R116 derivatives, viruses with an RSV-198nt insert are preferred oncolytic virus candidates for growth characteristics in eggs.

Figure 8 is a graph depicting the growth kinetics of r73T virus in veronol cells. The virus was also evaluated in serum-free vilo cell clone 51D11 (a proprietary cell line produced by medical immunology corporation (MedImmune)). All viruses replicated equally well under both moi conditions (0.001, up; 0.0001, down). The modification of 73TFPCs and the intergenic insertion in the HN-L junction of R116 did not affect virus growth in Vero cells.

Figures 9A-9D show that the r73T virus selectively replicates in tumor cells and is cytotoxic to tumor cells compared to non-neoplastic cells. Data were obtained after infection with r73T derivative at different doses ranging from 1 to 100,000PFU for 72 hours. Figure 9A is a graph showing the assessment of rT3T and its derivatives for cell killing in human fibrosarcoma HT1080 relative to untreated control cells. In HT1080 cancer cells, the virus with weakly virulent FPCS had the least killing, S116 at FPCS was moderate, and the virus with R116 at FPCS had as effective a cell killing as the R73T wt virus. FIG. 9B is a graph showing the targeting of normal human skin fibroblasts relative to untreated control cellsCell killing in cell CCD1122Sk cells, assessment of rT3T and its derivatives. In normal CCD1122SK cells, all viruses did not kill cells as effectively as cancer cells, with a reduction in killing efficiency of about 100-fold. Regardless of the FPCS sequence, all viruses have similar kills in normal cells, probably due to single-cycle replication (no viral transmission). FIG. 9C is a table showing cell killing efficiency in cancer cells and normal cells expressed as a 50% Effective Concentration (EC) extrapolated from dose-response curves using Prism 6.0₅₀). R116 derivatives with EC having 10PFU₅₀R73T wt possesses a similar EC₅₀Values, indicating that the modification of FPCS did not affect viral replication in cancer cells and cell killing efficiency. FIG. 9D is a graph showing viral replication in HT1080 and CCD1122SK cells at MOI0.01 at day 3 post infection. All viruses replicate preferentially in cancer cells but not in normal cells, with differences of about 1.5-2.0 logs.

Fig. 10A and 10B are graphs showing that the r73T derivative was effective in tumor regression when administered locally and systemically. To assess in vivo oncolytic activity, the HT1080 xenograft model was performed at 5x10⁶Cells/0.1 ml were formed by injecting HT1080 cells subcutaneously into 5-6 week old Balb/C athymic nude mice. FIG. 10A is a graph showing the effect of R116i-318-hGM-CSF administered intratumorally (it) or intravenously (iv). The data show that R116i-318-hGM-CSF derivatives have anti-tumor activity in vivo when delivered systemically or intratumorally to immunodeficient mice bearing human tumor xenografts. Tumor growth rates were compared between treatment and control groups. Tumor regression induced by both routes of administration was significantly different from the control group. When the tumor volume reaches about 65mm³When mice were randomly assigned to the indicated group (n-10). Mice received a single dose of Intratumoral (IT) administered PBS or 2x 10⁷Pfu R73T-hGM-CSF-R116i-198 or Intravenous (IV) administration by tail vein injection 1x10⁸PFU。*P<0.05, not paired student t-test. FIG. 10B is a diagram of 1X10 IV injection in HT1080 xenografts⁸PFU, the oncolytic activity of r73T derivatives was compared. Two doses of the r73T derivative were able to induce significant tumor regression with varying degrees of effect. r73T-lento was least effective in tumor regression, whereas r73T wt was most effective in tumor regression. r73T-lento has a similar effect as the S116 virus, although the S116 virus has 10-fold lower EC50 in cell killing in vitro (see fig. 9A). Up to day 9 after dose 2 (day 19 post tumor implantation), R73T-R116i-318 were as effective at inhibiting tumor growth as 73T wt, which again restored growth in R116i treated mice, but not in the 73T wt treated group. When the tumor volume reaches about 180mm³When the mice were randomly assigned to groups (n-7). Mice received PBS or 1x10 by IV administration⁸PFU R73T-hGM-CSF-lento (lento) or R73T-hGM-CSF-S116K113M114(S116KM) or R73T-hGM-CSF-R116i-318nt APMV-N (R116i) or R73T-hGM-CSF (R73 Ttw). Tumor size was measured every 3-4 days. P<0.05, not paired student t-test. The data show that the r73T derivative has anti-tumor activity in vivo when delivered systemically or intratumorally to immunodeficient mice bearing human tumor xenografts. Efficient cleavage of the F protein is important for viral replication in vitro and in vivo. Viruses with R116 at the FPCS are more effective in cell killing in vitro and in vivo.

FIGS. 11A-G depict the tissue biodistribution of r73T derivatives and the effect of mouse and human GM-CSF on tumor growth inhibition after intravenous delivery. To determine whether oncolytic NDV viruses selectively replicate in tumor tissue, viral clearance, viral distribution in different organs was determined. Will have a load of about 250mm³Size of subcutaneous HT1080 tumor in athymic nude mice at 1x10⁸Doses of PFU were treated intravenously with R116i (R73T-hGM-CSF-R116i-318ntAPMV-N) and on days 1, 4 or 8, and sacrificed (N ═ 3 per time point). Serum, lung, spleen, ovary and tumor were collected and the presence of virus was quantified. Viral replication in tumors and organs was assessed on days 1, 4 and 8 post infection. Figure 11A is a graph depicting quantification of virus in tissue by plaque assay in veronol cells. Virus was detected in organs only on day 1 (all onNone of the time points tested for virus in the ovaries, no data showed), and the virus load in tumor tissue was about 100-fold higher than in the lungs and spleen. The presence of the virus in the tumor persists for at least 8 days, indicating that the virus selectively replicates in the tumor tissue.

FIG. 11B is a graph depicting quantification of GM-CSF expressed by the virus in tissues as determined by ELISA for hGM-CSF transgene expression. Consistent with the viral replication data obtained by plaque assay, the level of hGM-CSF was highest and persisted in tumor tissue for more than 8 days. These data demonstrate that NDV virus replicates efficiently in tumor tissue and that the transgene is efficiently delivered to local tumor tissue.

FIG. 11C depicts graphs showing the effect of mGM-CSF expression on tumor growth inhibition in a HT1080 xenografted mouse tumor model. 5-6 week-old athymic nude mice in each group (seven mice per group) were treated with 5X10⁶Ht1080 cells were implanted subcutaneously (s.c.) in the right flank. When the tumor reaches 110mm³At volume of (6 days), tumors were given a single dose of 1x10⁸pfu rNDV, R116i-198RSV with mGM-CSF or hGM-CSF, were infected. Tumor size was measured every 3-4 days. R116i-198RSV with mGM-CSF was less effective in tumor growth inhibition than the hGM-CSF transgene. However, no difference in tumor growth inhibition was observed for S116 with hGM-CSF or mGM-CSF.

Figure 11D depicts graphs showing the effect of mGM-CSF on tumor-derived viral clearance in an HT1080 xenografted mouse tumor model. Athymic nude mice in each group (three per group) were treated with 5X10⁶Ht1080 cells were implanted subcutaneously (s.c.) in the right flank. When the tumor reaches 180mm³At volume of (10 th day), mice were dosed once at 1 × 10⁸pfu of R116i-198RSV or S116KM was treated intravenously. Tumors were collected on day 4 or 7 and virus titers in tumor tissues were determined by plaque assay. On day 4, R116i-198RSV and S116KM, with either mGM-CSF or hGM-CSF, both had comparable titers. On day 7, R116i-198RSV with mGM-CSF was greatly reduced compared to R116i-198RSV with hGM-CSF. In comparisonThe titers of S116mGM-CSF and hGM-CSF are comparable.

Figure 11E depicts a graph showing the effect of R116i-198RSV or S116KM infection on immune cell infiltration into tumors in an HT1080 xenograft mouse tumor model. Athymic nude mice in each group (three per group) were treated with 5X10⁶Ht1080 cells were implanted subcutaneously (s.c.) in the right flank. When the tumor reaches 180mm³At volume of (10 th day), mice were dosed once at 1 × 10⁸pfu of the indicated virus was treated intravenously. On day four, tumors were harvested and these tissues were treated by FACS analysis for neutrophil, NK cell and macrophage staining. R116i-198-RSV and S116-KM. R116i-198RSV with mGM-CSF has more immune cell infiltration.

Figure 11F depicts a table showing cytokine and chemokine upregulation in an HT1080 xenograft mouse tumor model. Athymic nude mice in each group (three per group) were treated with 5X10⁶Ht1080 cells were implanted subcutaneously (s.c.) in the right flank. When the tumor reaches 180mm³At volume of (10 th day), mice were dosed once at 1 × 10⁸pfu R116i-198RSV or S116KM with hGM-CSF or mGM-CSF was treated intravenously. On day four, tumors were harvested and these tissues were treated for cytokine and chemokine levels by Luminex analysis. In local tumor tissues, viral infection induces cytokine and chemokine production, the levels of which vary based on the viral backbone and human or mouse GM-CSF.

FIG. 11G depicts a graph showing that R116i-198RSV-hGM-CSF and R116i-318APMV-hGM-CSF are comparable in tumor growth inhibition in a HT1080 xenograft mouse tumor model. Athymic nude mice in each group (seven per group) were treated with 5X10⁶Ht1080 cells were implanted subcutaneously (s.c.) in the right flank. When the tumor reaches about 110mm³On volume (day 6), will be 1x10⁸A single dose of pfu was injected intratumorally with the virus. Tumor sizes were measured every 3-4 days and plotted. The insertion lengths of 198nt and 318nt did not affect the oncolytic activity of R116 i.

FIGS. 12A and 12B depict the construction of 73T antigenomic cDNA containing chimeric F and/or HN genes and their characteristics, and FIG. 12C compares the function of RNA polymerase complex activity. In chickens, viral surface glycoproteins are important antigens for immunogenicity and virulence. The F and/or HN genes of NDV are replaced by the corresponding extracellular (ecto) domains of other paramyxoviruses that are not virulent in chickens, alone or in combination. Parainfluenza virus 5(PIV 5) is a canine paramyxovirus and does not cause human disease, and pigeon paramyxovirus type 1 (PPMV-1) has been shown to be not virulent in chickens. FIG. 12A shows that the F and/or HN glycoprotein extracellular domains of the full-length antigenomic NDV73T cDNAs were replaced by those of PPMV-1 and/or PIV 5. NDV, PIV5, and PPMV-1 sequences are indicated with boxes colored blue, purple, or green, respectively. The length of the amino acids of the individual proteins or protein domains is indicated. FIG. 12B is a table showing the characterization of 73T derivatives containing chimeric F and/or HN genes. Plaque formation, relative HT1080 cell killing and MDT entry in vilo were performed as described previously. Chimeric viruses, except PVI-5F-HN, were recovered and grown in cells in the absence of exogenous trypsin. All three viruses formed considerable plaques and showed efficient cell-to-cell diffusion. PPMV-1F-HN or F chimeric viruses had MDT values of 79 and 84 hours, respectively, indicating potential virulence in chickens. Two PMI-1 chimeric viruses killed HT1080 cells efficiently at 71% and 61% levels. The PIV5-F chimera did not grow in egg and was not virulent in chicken, but killed HT1080 cells at 47%. Serum cross-reactivity between NDV and either the PPMV-1 or PIV5 chimeras was tested by neutralization assay using serum collected from mice given IV with 2 doses of 1x 108PFU R73T-R116 i. The r73T virus could be neutralized by NDV infected sera (titer 960), however the PPMV-1 and PIV5 chimeras were not neutralized (titer <4), confirming no cross-reactivity between NDV and either PPMV-1 or PIV 5. Chimeric viruses with different antigenicity can augment oncolytic viruses in patients who have developed an immune response against NDV during previous NDV therapy.

Figure 12C shows NDVRNA polymerase activity compared to other paramyxoviruses by minigenome assay. Using Lipofectamine 2000, T7 expressing cells were transfected with three plasmids expressing the NP, P, L proteins of NDV and a plasmid encoding the NDV antiminigenome cDNA (encoding the GFP gene), or a plasmid encoding the N, P, L gene of Measles Virus (MV) or Respiratory Syncytial Virus (RSV) and the respective RSV or MV GFP antiminigenome cDNA plasmid. Two or three days after transfection, minigenome replication was examined under a fluorescent microscope as indicated by expression of GFP. NDV has the strongest polymerase activity compared to measles virus and RSV.

Fig. 13A and B show the sensitivity of cancer cell lines to rNDV variants. Cancer cell lines from various source tissues were infected with NDV S116 or NDV R116i at an MOI of 0.1, and cell viability was assessed 72 hours post infection using the cell titer glo (celltitre glo). Sensitivity to NDV is defined as greater than 30% cell kill at 72 hours post infection. Figure 13A depicts a graph of the percentage of NDV sensitive human cancer cell lines in at least 16 broad indications. The number of cell lines in each group is indicated numerically and in the table. FIG. 13B depicts graphs depicting the sensitivity of 22 cancer cell lines to NDV variants. % cell kill was determined as the percentage of untreated control cells.

FIG. 14 is graphs showing cancer cell line versus recNDV^GM-CSFThe permissibility of (c). Cell killing of R116i-GM-CSF in representative tumor cell lines is presented in FIG. 14. The maximum% kill was determined 3 days after infection of the tumor cell line relative to the uninfected control at an MOI of 0.1. Although some cell lines are derived from the same tumor type, such as bladder, stomach, prostate, melanoma, breast, ovarian, pancreatic and lung cancers, they show different sensitivities to viral killing. The number of cell lines from each tumor indication, which can be identified by R116i-GM-CSF and so forth, is summarized in Table 1>The rate of 50% kills.

FIGS. 15A-F are graphs showing that NDV derivatives inhibit tumor growth in different mouse cancer models. FIGS. 15A and B are diagrams showing melanoma models in syngeneic micerecNDV in (B16F10AP3)^GM-CSFThe virus strain inhibits tumor growth. In a pilot study, 73T-R116i-hGM-CSF (FIG. 15A left) and R116i-mGM-CSF (FIG. 15A right) were evaluated for their oncolytic effects in the B16 melanoma model. Since the evaluation of virus tolerance was performed in B16 mice, the group was small in scale (n-3). Each virus was expressed at 2x 10⁷pfu was given intravenously (i.v) twice on days 11 and 14; at 2x 10⁷pfu was administered intraperitoneally (i.p) twice on days 11 and 14; or at 1.1x 10⁷pfu was administered intratumorally once on day 11 (i.t). The groups treated with R116-hGM-SCF or mGM-SCF by the three routes of administration had slower tumor growth rates compared to the untreated group. The tumor inhibition rate was statistically significant compared to the control group.

Figure 15B shows 1x10 administration into a tumor⁸Efficiency studies in the B16F10 syngeneic murine melanoma model of the S116NDV variant of pfu. Tumor growth inhibition is shown on the left panel, and individual animal measurements are shown on the middle panel, and survival plots are shown on the right.

Figure 15C shows that NDV has potent anti-tumor activity in an immunocompetent mouse CT26 colorectal tumor model. Specifically, fig. 15C provides for administration of 1x10 into a tumor⁸Efficiency studies in a CT26 syngeneic murine colorectal model of pfu encoding the S116NDV variant of human GM-CSF. Tumor growth inhibition is shown on the right panel and in the presence of H&IHC analysis of remaining tumors is shown on the right hand side of E and NDV staining. Indicating necrotic regions, evidence of multinuclear syncytia and active regions of the tumor.

FIGS. 15D-F depict tumor growth inhibition in an ovarian xenograft model. Athymic nude mice were implanted subcutaneously (s.c.) in the right flank with Ovcar4 cells. When the tumor reaches 100mm³At volume (v), mice were randomly assigned to treatment groups. The formed tumor is treated with 2.5x 10⁷pfu rNDV, with mGM-CSF or hGM-CSF R116i-318, was injected once a week. Tumor growth curves (fig. 15D) and histological analysis of tumors (fig. 15E and 15F) are shown.

Figure 16 is a table summarizing the characteristics of NDV constructs. Plaque formation in veronol cells without trypsin in the overlay after 36 hours incubation and visualized at 10-fold magnification. Cytotoxic effect of the virus on human fibrosarcoma HT1080 cells 72 hours post-infection at a multiplicity of infection (MOI) of 0.01. The relative percentage of viable cells was determined by comparing each sample to untreated cells considered to be 100% viable. The data presented in the table are relative percentages of dead cells. Pathogenicity of NDV in pathogen-free chickens at 1 day of age, expressed as the intracerebral pathogenicity index (ICPI). ICPI measurements were performed at the National Veterinary Service Laboratory (NVSL) (emms, iowa).

Figure 17 is a graph showing that NDV produced from egg and human cell lines exhibit different sensitivities to complement-mediated inactivation. Human serum (Sigma), st louis, missouri) with confirmed complement (C') activity was serially diluted with PBS and incubated with 100pfu of NDV for 1 hour at 37 ℃ before infecting vero cells for plaque assay. After incubation at 37 ℃ for 6 hours, plaques were visualized by gentian violet staining and scored. Viruses grown in chicken embryos are mostly inactivated by dilution with 1:10 to 1:40 serum. 293-grown virus is more resistant to C' than egg-grown virus, with approximately 40% infectivity retention at a serum concentration of 1: 40. However, the hela-grown virus is most resistant to C' mediated viral inactivation. 90% of live viruses are infectious at a serum concentration of 1: 40.

Fig. 18A and 18B are western blots showing a comparison of RCA protein levels in 293 and hela cells and in viruses produced from these two cell lines. For FIG. 18A, equal amounts of 293 and Hela S3 cells were loaded onto SDS-PAGE for Western blotting. Hela cells contained higher levels of hCD46, hCD55, and hCD59 proteins than 293 cells. Figure 18B shows western blots performed to examine the amount of protein in viruses from infected 293 or hela cells. Uninfected cells (mock) served as controls. Three CD molecules were detected in the virus from infected hela cells, at levels higher than those from 293 cells.

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Figure 19 shows the assessment of membrane-bound C 'regulatory factor (RCA) protein in C' mediated inactivation of virus. The cdnas encoding hCD55, hCD59, and hCD46 were synthesized from origanum (Origene) (rockville, maryland) or Genscript (pistavir, new jersey). Each gene cassette is inserted into the P-N intergenic region of the NDV antigenomic cDNA, and the recombinant virus is generated by reverse genetics. The recombinant virus was amplified in eggs and purified on a 15% -60% sucrose gradient and the viral band was pelleted by ultracentrifugation. Expression of recombinant NDV for each RCA protein was confirmed by western blotting.

Figure 20 is a diagram showing CD55 as the major RCA protein for preventing C' inactivation of NDV. NDV with hCD46, hCD55, or hCD59 were amplified in eggs, purified by sucrose gradient, incubated with human plasma diluted from 1:10 to 1:40 for 1 hour, and viral infectivity was examined by plaque assay in veronol cells. NDV produced in eggs with hCD55 was similarly resistant to C compared to NDV produced in hela cells, with about 65% active virus at a plasma concentration of 1:20 and about 80% active virus at a plasma concentration of 1: 40. hCD46 showed slight or slightly improved virus resistance to C' mediated inactivation, with about 20% more virus active when incubated with human plasma diluted 1:40 compared to the NDV control. No difference was detected at the lower plasma dilutions.

Detailed Description

The invention features compositions comprising attenuated newcastle disease virus and methods of using that virus for treating neoplasia.

The present invention is based, at least in part, on the discovery of oncolytic NDV's with reduced chicken virulence. As reported in more detail below, the NDV73T strain was derived from NDV MK-107, a commercial poultry vaccine first marketed in 1948 (mesovirulence). The NDV Mk-107 strain remained in the Ehrlich ascites tumor cells over 73 passages (Cassel et al, Cancer (Cancer), 7 months 1965; 18: 863-8). NDV Mk-107 was used in a series of PhI and Ph II clinical studies in the 70's of the 20 th century. NDV Mk-107 was also used as an immunotherapy in the 80's of the 20 th century to treat patients with advanced melanoma (Casel et al, cancer, 19831; 52: 856-.

To produce oncolytic NDV with reduced chicken virulence, the recombinant NDV73T virus strain includes certain genetic modifications. Specifically, the F protein cleavage sequence was changed, and the length of the HN-L intergenic sequence was increased. Advantageously, the modified virus can be used to express a transgene of interest. In one embodiment, the NDV73T strain comprises a transgene encoding a polypeptide that enhances the oncolytic properties of the recombinant NDV. In another embodiment, the NDV73T strain includes a transgene encoding a biomarker that provides a readout for monitoring viral replication. If desired, the NDV73T strain can be modified to incorporate additional genetic information that disrupts the normal transcriptional polarity of the standard genome and is expected to further reduce viral virulence in chickens. Thus, the present invention provides methods for using reverse genetically produced recombinant Newcastle Disease Virus (NDV) to reduce its pathogenesis in chickens while maintaining its selective cancer cell killing ability, and methods for producing the same. The invention also provides the construction and use of NDV as a viral vector to deliver and express heterologous gene products for enhanced cancer therapy. Described herein below are transgenes encoding exemplary therapeutic agents that can be delivered by NDV. In the working examples described herein below, the novel NDV viral construct expressing granulocyte macrophage colony stimulating factor (GM-CSF) selectively killed cancer cells, but did not kill normal cells. When tested in the xenograft HT1080 tumor model, cell killing was observed for this selectivity in multiple cancer cell lines as well as in vivo. The efficacy and selectivity of recombinant attenuated Newcastle Disease Virus (NDV) was also demonstrated in a melanoma model, where tumor regression was observed. In summary, the present invention provides for the insertion of one or more specific transgenes into a recombinant attenuated NDV vector, as well as for the efficient expression of the encoded protein in the tumor environment.

Newcastle disease virus

Newcastle Disease Virus (NDV) is an enveloped virus that comprises a linear, single-stranded, non-segmented, antisense RNA genome. The antisense, single-stranded genome of NDV encodes an RNA-directed RNA polymerase, a fusion (F) protein, a hemagglutinin-neuraminidase (HN) protein, a matrix protein, a phosphoprotein, and a nucleoprotein. The genomic RNA comprises the following sequence of genes: 3' -NP-P-M-F-HN-L. The construction of NDV RNA genomes is described in more detail herein below. The genomic RNA also contains a leader sequence at the 3' end.

The structural elements of the virion include the viral envelope, which is a lipid bilayer derived from the cytoplasmic membrane. The glycoprotein, hemagglutinin-neuraminidase (HN), protrudes from the envelope, allowing the virus to contain hemagglutinin and neuraminidase activities. The fusion glycoprotein (F), which is an integral membrane protein, is first produced as an inactive precursor and then cleaved post-translationally to produce two disulfide-linked polypeptides. This active F protein is involved in the invasion of NDV into host cells by promoting fusion of the viral envelope with the host cell plasma membrane. Matrix protein (M) is involved in viral assembly and interacts with viral membrane and nucleocapsid proteins.

The major protein subunit of the nucleocapsid is the Nucleocapsid Protein (NP) which confers helical symmetry on the capsid. Associated with the nucleocapsid are the P and L proteins. Phosphoproteins (P) which undergo phosphorylation are thought to play a regulatory role in transcription and may also be involved in methylation, phosphorylation and polyadenylation. For viral RNA synthesis, the L gene encoding RNA-dependent RNA polymerase is required along with the P protein. The L protein, which occupies nearly half of the coding capacity of the viral genome, is the largest of the viral proteins and plays an important role in transcription and replication.

Replication of all negative-strand RNA viruses, including NDV, is complicated by the lack of cellular machinery required to replicate the RNA. Furthermore, the negative strand genome cannot be directly translated into protein, but must first be transcribed into positive strand (mRNA) copies. Thus, when entering a host cell, genomic RNA alone cannot synthesize the desired RNA-dependent RNA polymerase. Upon infection, L, P and the NP protein must enter the cell along with the genome.

Without being bound by theory, it is hypothesized that most or all of the viral proteins that transcribe NDV mRNA also replicate. The mechanism by which other uses of the same complement of proteins are regulated (i.e., transcribed or replicated) is not yet clear. Following viral entry, transcription is initiated by the L protein using antisense RNA in the nucleocapsid as a template. Viral RNA synthesis is regulated such that it produces monocistronic mRNA during transcription. Following transcription, viral genome replication is the second event that occurs when a negative-strand RNA virus infects a cell. Like other negative-strand RNA viruses, viral genome replication of Newcastle Disease Virus (NDV) is mediated by virus-specific proteins. The first product of the replicated RNA synthesis is a complementary copy (i.e., plus polarity) of NDV genomic RNA (crna). These plus-stranded copies (antigenomes) differ from the plus-stranded mRNA transcripts in their terminal structure. Unlike mRNA transcripts, antigenomic cRNA is not capped and methylated at the 5 'end and is not truncated and polyadenylated at the 3' end. The cRNA is co-terminal with its negative-strand template and contains all the genetic information in each complementary form of the genomic RNA segment. The cRNA serves as a template for the synthesis of NDV minus-strand viral genomes.

Both NDV negative strand genome (vRNA) and antigenome (cRNA) are encapsidated by nucleocapsid proteins; the only non-encapsidated RNA species is viral mRNA. For NDV, the cytoplasm is the site of viral RNA replication, just as it is the site of transcription. Assembly of viral components may occur at the host cell plasma membrane. The mature virus is then released from the cells by budding.

Oncolytic virus

Viruses are known to exert oncolytic effects on tumor cells, and the use of oncolytic viruses as therapeutic agents has been reported. Some efforts have been made in treating cancer patients to use non-human viruses that exhibit moderate to high pathogenicity for their natural host. The present invention discloses methods for inducing tumor regression in human subjects using modified mesogenic strains of Newcastle Disease Virus (NDV) having a modified F protein cleavage site that are non-pathogenic (lentogenic) to poultry but exhibit oncolytic properties. The disclosed methods provide a safe, effective and reliable means to induce tumor regression in an individual in need thereof. These methods overcome the disadvantages of using pathogenic strains of the virus for human therapy.

Accordingly, in one aspect, the present invention provides a method for inducing tumor regression in a subject, the method comprising the step of administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a lentogenic oncolytic virus strain of NDV. According to one embodiment, the lentogenic oncolytic virus strain of NDV is NDV R73T-R116.

Oncolytic viruses are capable of exerting cytotoxic or killing effects on tumor cells in vitro and in vivo, with little or no effect on normal cells. The term "oncolytic activity" refers to the cytotoxic or killing activity of a virus that targets tumor cells. Without wishing to be bound by any mechanism of action, the oncolytic activity exerted by a low virulence viral strain of NDV (e.g., R73T-R116) may be primarily due to apoptosis and to a lesser extent to plasma membrane lysis, the latter with the release of active progeny into the cellular environment, subsequently infecting neighboring cells. Without wishing to be bound by a particular theory, it is believed that NDV has direct cytolytic activity on cancer cells. It is also believed that NDV is able to differentiate cancer cells specifically from normal, healthy cells. The results have indicated that several oncogenes (H-ras, N-ras, and N-myc) known to confer malignant behavior to cancer cells increase the sensitivity of the cells to killing by NDV. See lorentz (Lorence), r.m., Reichard, k.w., Cascino, c.j. et al, (1992), proceedings of the american society for cancer research (proc.am. assoc. cancer Res.), 33,398; reichard (Reichard), k.w., lorens, r.m., casinos, c.j., et al, (1992) surgical forum (surg. forum), 43, 603-. In addition, it has been observed that treatment of cells with retinoic acid (vitamin a) also increases the lysis of cancer cells by NDV. Licard, k.w., lorentz, r.m., karybigs (Katubig), b.b. et al, (1993), journal of pediatric surgery (j.pediatr.surg.), 28,1221.

Cytotoxic effects under in vitro or in vivo conditions can be detected by various means known in the art, for example, by inhibiting cell proliferation, by detecting tumor size using gadolinium enhanced MRI scanning, by radiolabeling tumors, and the like.

For clinical studies, it is desirable to obtain cloned viruses in order to ensure viral homology. The cloned virus may be produced according to any method available to the skilled person. For example, cloned viruses can be produced by limiting dilution or by plaque purification.

NDV culture

The viruses used in the present invention can be prepared by a variety of methods. For example, NDV may be prepared in fertilized eggs from 8 to 10 days old (available from SPAFAS, lornok, illinois). Methods for isolating viruses are known and described in the art, for example, by Wess (Weiss), S.R., and Bratt (Bratt), M.A. (1974) J.Virol (J.Virol), 13, 1220-. The process is further described in example #1 below. Using this separation method, NDV can be obtained in about 90% to 95% purity.

Alternatively, the virus may be produced in vitro cell culture. Preferably, the cell culture comprises mammalian cells, and more preferably, the cells can be used for virus production such as vilo cells. The virus will be purified by chromatography or other suitable method. These cells may be anchorage dependent or anchorage independent.

Cell culture techniques that can be used in the preparation of viruses are known in the art and may include the use of static culture flasks or roller flasks with large surface areas. Preferably, the type of culture system selected can support a relatively large number of cells. To produce large quantities of virus, a bioreactor process will be deployed, while cells are grown in microcarrier beads for virus infection and production.

Cell culture media that can be used in the production of viruses are known to those skilled in the art. The medium typically comprises a nutrient source, an antibiotic and albumin or a serum source containing one or more growth factors. The particular medium and components of the medium suitable for use in culturing the cells are selected within the skill of the art. In certain embodiments, trypsin is included in the growth medium. In other embodiments, trypsin is not included.

Culture conditions typically include incubation at a desired temperature (e.g., 37 ℃) in conjunction with selected concentrations of oxygen and carbon dioxide. The particular culture conditions selected can be determined based on the cells used in the culture, and the determination of such conditions is within the skill of the art.

Cells are placed in culture flasks and allowed to incubate and grow under selected culture conditions. Preferably, anchorage dependent cells are allowed to grow to confluence or peak growth. The time required for growth will vary depending on the amount of primary cell inoculum added to the flask and the doubling time of the cells used. Preferably, per cm²About 3X 10 of the floor³To about 3X 10⁵Cells and growth continued for one to five days. Virus inoculation for cell culture, media is removed from the cells (for adherent cells, by aspiration of culture media; for cells growing in suspension, by centrifugation of the cell suspension and aspiration of the cell supernatant) and the virus (after reconstitution) is added to the cells in a minimum volume of media or salt solution (such as Hank's balanced salt solution, Gibco) to prevent dehydration. Preferably, this volume ranges from about 10 to about 2500 microliters/cm²Flask surface area or 10⁵A cell. Preferred dilutions of the viral inoculum range from about 0.001 to about 10 infectious units per cell, with the optimum ratio depending on the particular virus and cell line. The virus then grows from 1 to 7 days, the length of time being determined primarily by the remaining viable cell lines. For NDV, the optimal harvest time is 1 to 5 days after virus inoculation.

The virus can then be obtained by: the supernatant is removed and replaced with fresh medium or fresh medium with fresh cells at intervals of 12 to 48 hours, or the virus is released in the supernatant by freeze-thawing the cells. The supernatant may then be centrifuged and ultracentrifuged to recover the virus in a relatively pure form or by chromatographic methods. The purity of the virus preparation can be tested by protein content assays and/or by electrophoresis. The virus may then be added to a pharmaceutically acceptable carrier as further described below.

Therapy method

Therapy may be provided anywhere cancer therapy is available: at home, a doctor's office, clinic, hospital clinic, or hospital. In one embodiment, the invention provides for the use of Newcastle Disease Virus (NDV) (e.g., R73T-R116).

Treatment is typically initiated at the hospital so that the physician can closely observe the effect of the therapy and make any needed adjustments. The duration of the therapy depends on the type of cancer to be treated, the age and condition of the patient, the stage and type of disease of the patient, and the physical response of the patient to the treatment. Administration can be at different intervals (e.g., daily, weekly, or monthly). Therapy may be given in intermittent cycles including rest periods so that the patient's body has the opportunity to build healthy new cells and regain strength.

Depending on the type of cancer and its stage of development, therapy may be used to slow cancer progression, slow cancer growth, kill or stop cancer cells that may spread from the original tumor to other parts of the body, alleviate symptoms caused by cancer, or prevent cancer in situ. Cancer growth is uncontrolled and progressive, and occurs under conditions that do not trigger or will cause cessation of the expansion of normal cells.

As noted above, treatment with the compositions of the invention can be combined with a therapy (e.g., radiation therapy, surgery, or chemotherapy) for the treatment of a proliferative disease, if desired.

Formulation of pharmaceutical compositions

Administration of a virus of the invention for treating a tumor (e.g., R73T-R116) can be by any suitable means, resulting in a therapeutic concentration that, in combination with other components, is effective to prevent, ameliorate, or reduce the tumor. The agent may be contained in any suitable carrier material in any suitable amount and is generally present in an amount of 1% to 95% by weight of the total weight of the composition. The composition may be provided in a dosage form suitable for parenteral (e.g., subcutaneous, intravenous, intramuscular, or intraperitoneal) administration. These pharmaceutical compositions may be formulated according to conventional pharmaceutical practice. (see, e.g., Remington: The Science and Practice of pharmacy (edition 20), eds., A.R. Thamnolo (Gennaro), Rispet Williams (Lippincott Williams) & Wilkins (Wilkins), 2000, and Encyclopedia of pharmaceutical technology, eds., J.Slaveck (Swarbick) and J.C. Boylan (Boylan), 1988-1999, Marcel Dekker, N.Y.).

The pharmaceutical compositions according to the invention may be formulated to release the active compound substantially immediately upon administration or at any predetermined time or period after administration. The latter type of composition, generally referred to as controlled release formulations, includes (i) formulations that produce a substantially constant concentration of drug in the body over an extended period of time; (ii) after a predetermined lag time, producing a substantially constant drug concentration formulation in the body over an extended period of time; (iii) formulations which maintain action over a predetermined period of time by maintaining a relatively constant effective level in the body whilst minimising concomitant undesirable side effects associated with fluctuations in the plasma level of the active (saw tooth kinetic pattern); (iv) formulations that act locally by, for example, placement of the controlled release composition adjacent to or in the space within the sarcoma; (v) formulations that allow for convenient administration, such that, for example, administration is once every one or two weeks; and (vi) formulations for delivering the therapeutic agent to sarcoma cells by targeting proliferating neoplastic cells using a carrier or chemical derivative. For some applications, controlled release formulations eliminate the need for frequent dosing during the day to maintain plasma levels at therapeutic levels.

Any of a number of strategies may be followed in order to obtain a controlled release wherein the release rate is greater than the metabolic rate of the compound in question. In one example, controlled release is achieved by appropriate selection of various formulation parameters and ingredients, including, for example, different controlled release compositions and coating types. Thus, the therapeutic agent is formulated with suitable excipients into a pharmaceutical composition that releases the therapeutic agent in a controlled manner upon administration. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, plasters, and liposomes.

The compositions of the present invention may be administered in unit dosage form in a pharmaceutically acceptable diluent, carrier, or excipient. Conventional pharmaceutical practice may be used to provide suitable formulations or compositions for administration of compounds to patients suffering from diseases caused by hyperproliferation of cells. Administration may begin before the patient is symptomatic.

Any suitable route of administration may be used, for example, administration may be parenteral, intravenous, intraarterial, subcutaneous, intratumoral, intramuscular, intracranial, intraorbital, ocular, intraventricular, intrahepatic, intracapsular, intrathecal, intracisternal, intraperitoneal, intranasal, spray, suppository, or oral administration. For example, the therapeutic formulation may be in the form of a liquid solution or suspension; for oral administration, the formulation may be in the form of a tablet or capsule; and for intranasal formulations, in the form of powders, nasal drops, or aerosols. For any of the methods of the above-mentioned applications, the compositions of the present invention are preferably administered or applied intravenously (e.g., by injection) to the site of the desired apoptotic event.

Methods well known in the art for preparing formulations are found, for example, "remington: pharmaceutical technology and Practice (Remington: The Science and Practice of pharmacy) "editor, A.R. Eunaro (Gennaro), Riping Kort Williams (Lippincott Williams) & Wilkins (Wilkins), Philadelphia, Pa., 2000. Formulations for parenteral administration may, for example, comprise excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated naphthalenes. Biocompatible, biodegradable lactide polymers, lactide/glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for delivering agents include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may include excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops or as a gel.

The formulations can be administered to a human patient in a therapeutically effective amount (e.g., an amount that prevents, eliminates, or reduces the pathological state) to provide therapy for a disease or disorder. The preferred dosage of the compositions of the invention may depend on variables such as the type and extent of the disorder, the overall health status of the particular patient, the formulation of the compound excipients, and the route of administration thereof.

Human dosages for any of the therapies described herein can be initially determined by extrapolation from the amount of compound used in mice, as one skilled in the art will recognize, and it is routine in the art to modify dosages for humans as compared to animal models. In certain embodiments, it is contemplated that the dose may be from about 10⁷pfu to about 10¹¹Between pfu; or from about 10⁸pfu to about 10¹⁰pfu or from about 10⁹pfu to about 10¹¹pfu was varied. In other embodiments, the dose may be about 10⁷pfu、10⁸pfu、10⁹pfu、10¹⁰pfu、10¹¹pfu. Of course, as is conventional in such treatment regimens, the dosage may be adjusted up or down, depending on the initial clinical trial and the needs of the particular patient.

Selection of treatment methods

After the subject is diagnosed with a neoplasia, a treatment method is selected. In neoplasia, for example, multiple standard therapy regimens are available. The marker profile of neoplasia is used in the selection of treatment methods. In one embodiment, the neoplastic cells respond to cell killing by NDV (e.g., R73T-R116).

Less aggressive neoplasias may be sensitive to conservative treatment approaches. More aggressive neoplasias (e.g., metastatic neoplasias) are less sensitive to conservative treatment methods and may relapse. When the method of the invention indicates that the neoplasia is very aggressive, an aggressive treatment should be selected. Aggressive treatment regimens typically include one or more of the following therapies: surgical resection, radiation therapy, or chemotherapy.

Assay for measuring cell viability

Agents (e.g., NDV) useful in the methods of the invention include those that induce neoplastic cell death and/or reduce neoplastic cell survival, i.e., viability.

Assays for measuring cell viability are known in the art and are described, for example, by clauchi (Crouch) et al (journal of immunology. meth.), 160, 81-8; kangas et al (Med. biol.)62,338-43, 1984); lundin et al, (methods in enzymology 133,27-42,1986); petty (Petty) et al (Comparison of journal of bioluminescence and chemiluminescence (Comparison of j. biolum. chemillum.) 10,29-34, 1995); korui (Cree) et al (AntiCancer Drugs)6:398-404, 1995). Cell viability can be determined using a variety of methods, including MTT (3- (4, 5-dimethylthiazolyl) -2, 5-diphenyltetrazolium bromide) (Barltop, Bioorganic and pharmaceutical chemistry bulletin (Bioorg).&Med.chem.lett.)1:611,1991; krey et al, Cancer Comm 3,207-12, 1991; puro (Paull), journal of heterocyclic chemistry (j. heterocyclic Chem.)25,911,1988). Assays for cell viability are also commercially available. Such assays include, but are not limited toA luminescent cell viability assay (Promega) that uses luciferase technology to detect ATP and quantify the health and number of cells in culture, anda luminescent cell viability assay, which is a Lactate Dehydrogenase (LDH) cytotoxicity assay (promegate).

Candidate compounds that induce or increase neoplastic cell death (e.g., increase apoptosis, decrease cell survival) may also be useful as anti-tumor therapies. Assays for measuring apoptosis are known to the skilled person. Apoptotic cells are characterized by characteristic morphological changes including chromatin condensation, cell shrinkage, and membrane blebbing, which can be clearly observed using light microscopy. Biochemical features of apoptosis include DNA fragmentation, protein cleavage at specific sites, increased mitochondrial membrane permeability, and the presence of phosphatidylserine on the cell membrane surface. Assays directed to apoptosis are known in the art. Exemplary assays include TUNEL (terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling) assays, caspase activity (specifically caspase-3) assays, and assays directed to fas ligand and annexin V. Commercially available products for detecting apoptosis include, for example,homogeneous caspase-3/7 assay, FragEL TUNEL kit (ONCOGENE RESEARCH PRODUCTS, Inc. (Sone RESEARCH PRODUCTS), san Diego, Calif.), ApoBrdU DNA fragmentation assay (bioVISION, mountain City, Calif.), and DNA ladder detection kit for apoptosis (bioVISION, mountain City, Calif.).

Neoplastic cells have a tendency to metastasize or spread from their initial location to distant points throughout the body. Assays directed to metastatic potential or invasiveness are known to the skilled person. Such assays include in vitro assays for loss of contact inhibition (Kim et al, Proc Natl Acad Sci U S A101: 16251-plus 6,2004), increasing soft agar colony formation in vitro (Bell (Zhong) et al, J International Oncology (Int J Oncol.)24(6): 1573-plus 9,2004), the lung metastasis model (Datta) et al, in vivo, 16: 451-plus 7,2002) and matrigel-based cell invasion assays (Hagemann et al, carcinogenesis, 25: 1543-plus 1549, 2004). In vivo screening methods for cell invasiveness are also known in the art and include, for example, tumorigenic screening in athymic nude mice. A common in vitro assay to assess metastasis is a matrigel-based cell invasion assay (BD Bioscience, franklin lake, new jersey).

Candidate compounds selected using any of the screening methods described herein are tested for their efficacy, if desired, using an animal model of neoplasia. In one embodiment, mice are injected with neoplastic human cells. Mice containing neoplastic cells are then injected daily (e.g., intraperitoneally) with vehicle (PBS) or candidate compound for an empirically determined period of time. The mice are then euthanized and tumor tissue collected and analyzed for the levels of NDV, NDV polypeptide, and/or NDV marker (e.g., a transgene encoding a detectable moiety) using the methods described herein. A compound that decreases the expression of NDV, NDV polypeptide, or NDV marker level mRNA or protein relative to a control level is expected to be effective in treating a tumor in a subject (e.g., a human subject). In another example, the effect of a candidate compound on tumor burden is analyzed in mice injected with human neoplastic cells. Neoplastic cells are allowed to grow to form clumps quickly. Mice were then treated daily with candidate compound or vehicle (PBS) for an empirically determined period of time. Mice were euthanized and tumor tissue was collected. The mass of tumor tissue in mice treated with the selected candidate compound is compared to the mass of tumor tissue present in corresponding control mice.

Reagent kit

The invention provides kits for treating or preventing sarcoma. In one embodiment, the kit comprises a therapeutic or prophylactic composition comprising an effective amount of NDV (e.g., R73T-R116) in a unit dosage form. In another embodiment, the kit comprises a therapeutic or prophylactic composition comprising an effective amount of NDV (e.g., R73T-R116) in a unit dosage form.

In some embodiments, the kit comprises a sterile container containing the therapeutic or prophylactic composition; such containers may be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister packs, or other suitable container forms known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil, or other material suitable for containing a medicament.

If desired, the antibodies of the invention are provided with instructions for administering an NDV (e.g., R73T-R116) to a subject having or at risk of developing a neoplasia. The instructions will generally include information regarding the use of the composition for treating or preventing neoplasia. In other embodiments, the instructions include at least one of: description of therapeutic agents; a dosage schedule and administration for the treatment or prevention of neoplasia or symptoms thereof; matters to be noted; warning information; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical studies; and/or references. These instructions may be printed directly on the container (when present), or applied to the container as a label, or as individual pages, brochures, cards, or folders provided in or with the container.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of those skilled in the art. Such techniques are well explained in the literature, such as "molecular cloning: a laboratory Manual (Molecular Cloning: A laboratory Manual), second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" (Methods in Enzymology) "A Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for mammalian cells (Gene Transfer Vectors for Mammaliancells)" (Miller and Calos, 1987); "Current protocols in molecular Biology" (Ausubel, 1987); "PCR: polymerase chain reaction (PCR: The Polymerase chain reaction), (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention and, as such, are contemplated for use in the preparation and practice of the invention. Techniques that are particularly useful for particular embodiments are discussed in the following sections.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to prepare and use the tests, screens, and therapeutic methods of the present invention, and are not intended to limit the scope of what the inventors regard as their invention.

Examples of the invention

Example 1. assembly of antigenomic cDNA of NDV strain 73T.

Six subgenomic cDNA fragments generated by high fidelity RT-PCR were assembled into the pUC19 vector. The full-length cDNA of NDV73T was designated p 73T. The nucleotide and deduced amino acid sequence of the F Protein Cleavage Site (FPCS) in 73T was modified to the sequence of glycoprotein B (gB) of NDV LaSota virus strain (lento) or Cytomegalovirus (CMV) (S116) (FIG. 1; double-slanted lines indicate cleavage sites for the F protein). The cDNA was completely sequenced to confirm the viral sequence. F protein cleavage site: wt: ggg agg aga cag aaa cgc ttt, respectively; lento: ggg ggg aga cag gaa cgc ctt, respectively; s116: cat aataga acg aaa tcc ttt, respectively; s116 KM: cat aat aaa atg aaa tcc ttt, respectively; r116: cat aat aga acgaaa cgc ttt are provided.

Example 2 transgene insertion into NDV73T genome.

The transgene was inserted into p73T at two positions: the intergenic sequence between P or M or between HN and L junctions (FIG. 2A). To insert a single transgene cassette at the P-M or HN-L junction, construction of the P73T cDNA containing the transgene at the P-M or HN-L junction was performed by inserting the transgene cassette into the AfeI site created between the P and M genes (nt3148) or between the HN and L genes (nt 8231). The inserted gene cassette comprises the gene ends (GE; 5' -TTAAGAAAAA)A-3'), intergenic nucleotides (T), gene promoter sequences (GS; 5'-ACGGGTAGA-3') and transgenic open reading frames(ORF). In addition, ten nucleotides (5'-cgccgccacc-3') were inserted upstream of the start site to introduce the Kozak sequence. The full-length (FL)73T cDNA containing the transgene at the P-M or HN-L junction was designated P73T-P1 or P73T-HN1, respectively. A full-length cDNA containing two separate transgenes at the junction of P-M and HN-L in a single genome was constructed and designated P73T-P1-HN1 (FIG. 2B). To insert both transgene cassettes at the same junction (e.g., P-M), an AfeI site was introduced at the end of the ORF of the first transgene (#1) (nt 3169). The 2 nd transgenic ORF was PCR amplified using primers containing GE and GS sequences and inserted at the AfeI site. The antigenomic cDNA containing two transgene cassettes at the P-M junction was designated P73T-P2.

Example 3 recovery of infectious recombinant NDV strain 73T with modified FPCS (r 73T).

NDV73T NP, P, L and antigen cDNA (p73T-lento or p73T-S116) were cloned under the control of a T7RNA polymerase promoter and terminator. The four plasmids were co-transfected into RNA polymerase expressing cell lines (fig. 3A). The recovered virus was designated r73T-lento or r 73T-S116. R73T-lento and r73T-S116 were passaged in Vero cells with media with or without trypsin supplementation. Growth of r73T-lento is trypsin dependent, however r73T-S116 can be grown in medium without trypsin supplementation. F Protein Cleavage Sequence (FPCS) in r73T-S116 was evaluated with or without hGM-CSF at the P-M junction. The 73T-S116 virus strain was further passaged for 10 passages in vilo and human fibrosarcoma HT1080 cells at MOI0.01 in medium without trypsin supplementation. Mutations occur in FPCS (R113K and/or Q114M) at generation 7. The S116R mutation was detected at passage 9. At passage 10, F, HN and the transgene were sequenced and no additional mutations were found.

Example 4 characterization of recombinant 73T virus strains with different F protein cleavage sequences.

A recombinant 73T virus strain with a novel modified F Protein Cleavage Sequence (FPCS) includes the following sequences:

-Lento：¹¹¹G-G-R-Q-E-R/L-I¹¹⁸

-S116：¹¹¹H-N-R-T-K-S/F¹¹⁷

-S116K：¹¹¹H-N-K-T-K-S/F¹¹⁷

-S116M¹¹¹H-N-R-M-K-S/F¹¹⁷

-S116KM¹¹¹H-N-K-M-K-S/F-I¹¹⁸

-R116：¹¹¹H-N-R-T-K-R/F-I¹¹⁸

recombinant 73T virus strains with different FPCS were characterized for MDT, ICPI, relative HT1080 cell killing, replication in vilo cells, and replication in eggs (fig. 4).

Avian virulence of NDV is determined primarily by the F Protein Cleavage Sequence (FPCS). R73T-lento was engineered to contain FPCS of the non-virulent strain LaSota. Replication of the LaSota virus in tissue culture is trypsin dependent, since the F protein cannot be cleaved. In vero cells without trypsin supplementation, r73T-lento formed tiny plaques, indicating that the F protein was not cleaved and that the virus was not able to spread efficiently from cell to cell. rR73-lento replicates at low levels in Villolo cells (7.5X 10)³pfu/ml), but replicate efficiently (5.7x 10) in eggs with endogenous trypsin-like enzymes⁸pfu/ml). Such as the mean time to death (MDT) of embryos inoculated with virus (MDT)>156 hours) and by intracerebral pathogenicity index (ICPI; ICPI ═ 0.00), r73-lento was not virulent in chickens and had low cytotoxicity in HT1080 cells (13% cell kill).

r73T-S116 can form relatively large plaques and reach 4.4x10 in Vero cells⁶Titer of pfu/ml. This titer was compared to the titer obtained when r73T-S116 was grown in vilo cells supplemented with trypsin. This data indicates that the Fusion Protein Cleavage Site (FPCS) of r73T-S116 can be cleaved in tissue culture in the absence of exogenous trypsin. It is not virulent in chickens and showed 31% cell killing in HT1080 cells. Genetic stability against r73T-S116 was tested by in vitro cell passaging.

After 10 passages in vilo or HT1080 cells, the following amino acid substitutions were found in FPCS: R113K, Q114M, and/or S116R. To eliminate the possibility of additional sequence changes occurring in the viral genome, recombinant r73T-S116 mutant viruses were constructed by reverse genetics and evaluated. Except for R73T-R116, R73T-S116 and its derivatives are similar to the parent S116, as these mutant viruses are not virulent in chickens and are capable of a similar level of HT1080 cell killing. Ht1080 cell killing was between 29% -31% for single mutations and 48% for double mutations.

Plaque sizes for M114 and K113M114 were significantly larger than S116. The 73T-R116 mutation results in an amino acid change at residue 116(S116R) in FPCS. R116 next to the cleavage site is known to be very important for efficient cleavage of the F protein. R73T-R116 formed large plaques in vilo cells, grew to similar titers with or without trypsin supplementation, and effectively killed HT1080 cells (80%). R116 increased chicken virulence as shown by MDT assay (72, 80 hours). Although the ICPI value (0.65) was <0.7 in one test, it preferably further reduced its chicken virulence.

Example 5.R73T-R116 derivatives have reduced chicken virulence.

The virus can be engineered to express a transgene at the P-M junction (1), a transgene at the HN-L junction (2), and an enlarged HN-L intergenic region extended by insertion of a non-coding sequence (3) (FIG. 5A). The same design of FIG. 2A incorporating the transgene cassette at the P-M junction is used herein. The second transgene cassette comprises the L gene promoter sequence (GS; 5' -ACGGGTAGA-3'), the Open Reading Frame (ORF) of the transgene, sequences from the 3' untranslated region of the L gene (italics) and the L gene end sequence (GE; 5'-TTAAGAAAAAA-3'). The non-coding sequences for increasing the HN-L junction region were taken from paramyxovirus type 1 (APMV-1), Respiratory Syncytial Virus (RSV) or random sequences without homology to any known sequence. The insertion may be in the range of 60-318 nt. Insertion of a second transgene at HN-L allows the virus to express both transgenes, e.g., hGM-CS and GFP. Sequences of insertions at the HN-L junction are listed (FIG. 5B).

Example 6 modification of R73T-R116 and characterization of R73T-R116 derivatives.

To reduce R73T-R116 virulence in chickens, the R73T-R116 virus was modified to increase the HN and L intergenic sequences by inserting sequences of various lengths. R73T-R116 derivatives were evaluated for: infectivity, by examining plaque formation and replication in cells and eggs; avian pathogenicity, by testing for MDT and ICPI; and tumor cell killing (fig. 6A).

Intergenic insertions of 318nt from APMV, 198nt from RSV and 198 random sequences did reduce virulence in chickens, accompanied by MDT>156 hours and ICPI of 0.27, 0.0375 and 0, respectively. Long insertions (198 nt randomized) had an increased effect on reducing virulence compared to short insertions (60 nt randomized). Insertions 144, 102 and 60nt were somewhat virulent in chickens, but the MDT time was shorter. The ICPI values for inserts 144, 102, and 60nt are 0.74, 0.51, and 0.78, respectively. Insertion of the avirulent sequence (random 198nt) is more effective in reducing virulence than insertion of the viral sequence (RSV-198 nt). Insertion of the 2 nd transgene cassette (EGFP) at the HN-L junction did not reduce chicken virulence (ICPI at MDT 86 hours and 0.82). All insertions slightly reduced virus replication in eggs by up to 4-fold, but did not affect virus replication in Vero cells (about 10)⁶pfu/ml). Although the mutant virus was more attenuated in chickens, the tumor cell killing function was not affected. All R73T-R116 derivatives had tumor cell killing efficiencies ranging from 75% to 86% at day 3 of infection.

Example 7. growth kinetics of r73T virus in egg and vilo cells.

To determine the growth conditions for the r73T virus, growth kinetics studies were performed in egg (fig. 7) and vilo cells (fig. 8). The chicken embryos are infected with the indicated virus at 100, 1000, 10,000 or 100,000 FFU/egg and incubated for 2,3 or 4 days. Allantoic fluid was obtained and virus titer was determined by FFA. As shown in fig. 7, 100 FFU/egg had low titer at day 1, but peak titer was reached at day 2. In summary, most viruses reached a peak titer of about 8logs/mL on day 2, regardless of the dose used for vaccination. R116i-198 had the lowest titer, with low inoculum size yielding the highest yield on day 2, indicating the possible accumulation of defective particles. The R116 i-318-APMV-insertion also had a lower peak titer of about 7 logs. R116i-198-RSV reached a peak titer of 7.5 logs. S116, constructed by reverse genetics, did not reduce virus production in eggs. Thus, among the R116 derivatives, viruses with an RSV-198nt insert are preferred oncolytic virus candidates for growth characteristics in eggs.

The virus was also evaluated in serum-free vilo cell clone 51D11 (a proprietary cell line produced by medical immunology corporation (MedImmune)). All viruses replicated well under both MOI conditions (0.001 and 0.0001) (fig. 8). The modification 73T FPCS and intergenic insertion in the HN-L junction of R116 did not affect virus growth in Vero cells.

Example 8 selective cell killing of r73T or a derivative in cancer cells compared to normal cells.

Cell killing of rT3T and its derivatives in human fibrosarcoma HT1080 was assessed relative to untreated control cells compared to normal human skin fibroblasts CCD1122Sk cells (fig. 9A and 9B). Data were obtained after infection with r73T derivative at different doses ranging from 1 to 100,000PFU for 72 hours. In HT1080 cancer cells, the virus with the attenuated FPCS had the least killing, S116 at PCS was moderate, and the virus with R116 at FPCS had as effective a cell killing as the R73T wt virus. In normal CCD1122SK cells, all viruses did not kill as efficiently as cancer cells. The reduction in killing efficiency is about 100 times. Regardless of the FPCS sequence, all viruses have similar killing efficiency in normal cells. Cell killing efficiency in cancer and normal cells was expressed as a 50% Effective Concentration (EC) extrapolated from dose-response curves using Prism 6.0₅₀) (FIG. 9C). EC of R116 derivative with 10PFU₅₀R73T wt has a similar EC₅₀Values, indicating that the modified FPCS did not affect viral replication in cancer cells and cell killing efficiency. Replication of these viruses in HT1080 and CCD1122SK cells was performed at MOI0.01 as determined on day 3 post-infection (fig. 9D). All viruses preferentially appear in cancerReplication in cells beyond normal cells is accompanied by a difference of about 1.5-2.0 logs.

Example 9 r73T derivatives have in vivo anti-tumor activity when delivered systemically or intratumorally to immunodeficient mice bearing human tumor xenografts.

To assess in vivo oncolytic activity, the HT1080 xenograft model was performed at 5x10⁶Cells/0.1 ml were formed by injecting HT1080 cells subcutaneously into 5-6 week old Balb/C athymic nude mice. Since hGM-CSF is not cross-reactive in mice, this study was not suitable for evaluating the transgenic effect, but for evaluating the oncolytic capacity of the various r73T constructs. R116-318i-hGM-CSF was administered intratumorally (it) or intravenously (iv) and tumor growth rates were compared between the treated and control groups (FIG. 10A).

When the tumor volume reaches about 65mm³When mice were randomly assigned to the indicated group (N ═ 10). Mice received a single dose of Intratumoral (IT) administered PBS or 2x 10⁷Pfu R73T-hGM-CSF-R116i-198 or by tail vein injection via Intravenous (IV) administration of 1x10⁸PFU. Tumor size was measured every 3-4 days. As presented in fig. 10A, R73T-R116i can induce significant tumor regression when administered IV or IT. The number of tumor regressions induced by the two pathways was comparable and significantly different from the control group.

After 1X10 injection by IT⁸The oncolytic activity of r73T derivatives was compared in PFU of HT1080 xenografts. When the tumor volume reaches about 180mm³When the mice were randomly assigned to groups (n-7). Mice received PBS or 1X10⁸PFU R73T-hGM-CSF-lento (lento) or R73T-hGM-CSF-S116K113M114(S116KM) or R73T-hGM-CSF-R116i-318nt APMV-N (R116i) or R73T-hGM-CSF (R73T wt). Tumor size (. about.P) was measured every 3-4 days<0.05, unpaired student t-test). Two doses of the r73T derivative were able to induce significant tumor regression, although there was a difference in the effect. r73T-lento was the least effective, while r73T wt was the most effective in tumor regression. r73T-lento has a similar effect as S116 virus, although S116 virus has 10-fold lower EC in cell killing in vitro₅₀(FIG. 9A). Up to day 9 after dose 2 (day 19 post tumor implantation), R73T-R116i-318 were as effective at inhibiting tumor growth as 73T wt, which again restored growth in R116i treated mice, but not in the 73T wt treated group. These data show that the r73T derivative has anti-tumor activity in vivo when delivered systemically or intratumorally to immunodeficient mice bearing human tumor xenografts. Efficient cleavage of the F protein is important for viral replication in vitro and in vivo. Viruses with R116 at the FPCS are more effective in cell killing in vitro and in vivo.

Example 10 tissue biodistribution of r73T derivatives after intravenous delivery.

To determine whether oncolytic NDV viruses selectively replicate in tumor tissue, viral clearance, viral distribution in different organs was determined. Will have a load of about 250mm³Size of subcutaneous HT1080 tumor in athymic nude mice at 1x10⁸Doses of PFU were treated intravenously with R116i (R73T-hGM-CSF-R116i-318nt APMV-N) and sacrificed on days 1, 4 or 8 (N ═ 3 per time point). Serum, lung, spleen, ovary and tumor were collected.

The presence of virus was quantified by plaque assay in veronol cells and the expression of hGM-CSF transgene was measured by ELISA assay. Viral replication in tumors and organs was assessed on days 1, 4 and 8 post infection. Viruses were detected in organs only on day 1 (no virus in ovaries was detected at all time points) and the load of virus in tumor tissue was about 100-fold higher than in lungs and spleen (fig. 11A). The presence of the virus in the tumor persists for at least 8 days, indicating that the virus selectively replicates in the tumor tissue. Consistent with the viral replication data, the level of hGM-CSF was highest and lasted for more than 8 days (fig. 11B). These data demonstrate that NDV virus replicates efficiently in tumor tissue and that the transgene is efficiently delivered to local tumor tissue.

Example 11 antigenomic cDNA of 73T comprising chimeric F and/or HN genes.

In chickens, viral surface glycoproteins are important antigens for immunogenicity and virulence. Strategies were explored to replace the F and/or HN genes of NDV by the corresponding extracellular (ecto) regions of other paramyxoviruses that are not virulent in chickens, alone or in combination. Parainfluenza virus 5(PIV 5) is a canine paramyxovirus and does not cause disease in humans. As previously reported, it has been shown that pigeon paramyxovirus type 1 (PPMV-1) is not virulent in chickens with an ICPI of 0.025 and is antigenically different from NDV (Dortmans et al, veterinary microbiology (Veterinary microbiology), 2010, Vol.143, p.139- & 144). There are two genetically closely related variants of pigeon paramyxovirus type 1 (PPMV-1) with the same velogenic fusion protein cleavage site, but with strongly contrasting virulence (veterinary microbiology, 2010, 143: 139-144). Full-length antigenomic cDNA of NDV73T was generated, in which the F and/or HN glycoprotein extracellular domains were exchanged with PPMV-1 and/or PIV5 (fig. 12A). NDV, PIV5, and PPMV-1 sequences are indicated with boxes colored blue, purple, or green, respectively.

The length of the amino acids of the individual proteins or protein domains is indicated. Plaque formation, relative HT1080 cell killing and MDT in vilo proceeded as described previously (fig. 12B). Chimeric viruses, except PIV-5F-HN, were recovered and were able to grow in cells in the absence of exogenous trypsin. All three viruses formed considerable plaques, showing efficient cell-to-cell diffusion. PPMV-1F-HN or F chimeric viruses had MDT values of 79 and 84 hours, respectively, indicating potential virulence in chickens. Both PMI-1 chimeras killed HT1080 cells effectively at 71% and 61% levels. The PIV5-F chimera did not grow in egg and was not virulent in chicken and killed HT1080 cells at 47%. Serum cross-reactivity between NDV and PPMV-1 or PIV5 chimeras was tested by neutralization assay using serum collected from mice that received 2 intravenous doses of 1x10⁸R73T-R116i of PFU. The r73T virus was neutralized with NDV-infected serum (titer 960), but the PPMV-1 and PIV5 chimeras were not neutralized (titer 960)<4). This result confirmed that there was no cross-reactivity between NDV and either PPMV-1 or PIV 5. Against the development of anti-NDV immunity in the course of treatment prior to NDVPatients with an immune response, chimeric viruses with different antigenicities have the potential to act as elevated oncolytic viruses.

NDVRNA polymerase activity was compared to other paramyxoviruses by minigenome assay (fig. 12C). Using Lipofectamine 2000, T7 expressing cells were transfected with three plasmids expressing the NP, P, L proteins of NDV and a plasmid encoding the NDV antiminigenome cDNA (encoding the GFP gene), or a plasmid encoding the N, P, L gene of Measles Virus (MV) or Respiratory Syncytial Virus (RSV) and the respective RSV or MV GFP antiminigenome cDNA plasmid. Two or three days after transfection, minigenome replication was examined under a fluorescent microscope as indicated by expression of GFP. NDV has the strongest polymerase activity compared to measles virus and RSV.

Example 12 cancer cells sensitive to NDV are identified by a cytometric screen.

To understand what tumor types may be susceptible to NDV oncolytic effects, 180 cancer cell lines and indications that would encompass a wide range of tumor types were tested for sensitivity to recombinant NDV and variants thereof. Cell lines were obtained from the american type tissue collection (manassas, virginia), or the european collection of cell cultures (ECACC), and were cultured in media and under conditions suggested by the supplier. 10,000 cancer cell lines were seeded on 96-well plates and infected with virus 6 hours later. The virus concentration ranged from MOI 10-0.0001 (or 1 to 100,000 pfu/well). Cell viability was determined 48-96 hours post infection. The cutoff of > 30% cell kill was used to determine sensitivity 72 hours after viral infection with an MOI of 0.1. Fig. 13A and 13B provide an overview of sensitivity by tumor type. Hematologic cancer cell lines (leukemias and lymphomas) are relatively insensitive to NDV oncolytic effects, whereas most melanoma, ovarian and pancreatic cell lines tested are sensitive to NDV. Approximately 58% of all human cancer cell lines tested were sensitive to NDVR116i at the FPCS, relative to 4% of the cell lines tested being sensitive to S-based viruses. This is most likely due to cleavage of the F protein by proteases. The R116i virus is more likely to be cleaved by the ubiquitous furin-like protease, whereas proteases that are likely to cleave the S116F protein are undefined and may not be widely expressed. The re-derived S116-KM variants resulted in larger plaque sizes and improved oncolytic capacity relative to the original S116 variant. To test this additional panel of 22 cancer cell lines, which have been determined to have a range of sensitivity to NDV, R116 viruses were infected with GFP-expressing variants and cell viability was determined 72 hours post-infection. Using the same sensitivity cut-off as described above, 41% of the tested cell lines were sensitive to R116i, 4% were sensitive to S116, and the re-derived S-KM was more potent than S116NDV, and 27% were sensitive to S116 NDV.

TABLE 4 summary of cancer cell susceptibility to viral killing by R116-hGM-CSF

Cells derived from the indicated cancer tissues were tested for cell killing using recombinant NDV73T with R116 at FPCS and human GM-CSF. The number of cells showed greater than 50% killing by viral infection at a moi of 0.1 and is indicative of the total cell line screened.

Example 13 in a syngeneic melanoma model, r73T derivatives have tumor killing and/or tumor growth inhibiting activity.

Following tumor model improvement, S116-RD NDV encoding human or murine GM-CSF was tested for efficacy in an improved B16F10 syngeneic model (FIGS. 15A and 15B). Intratumoral infection 1x10⁸pfu was continued for 3 doses and a minimum of 8 mice were present per group. By using>Tumor growth inhibition of 80% confirmed significant tumor growth inhibition (fig. 15B). Short-term tumor regression was achieved using repeated intratumoral (i.t.) doses, which also resulted in a significant increase in survival time, relative to 42 days in the treated group, with 18 days in the control group. Treatment was stopped after 3 doses, and tumors subsequently re-grew. At day 50, individual animals with no evidence of residual tumor in the S116-mGM-CSF group were again challenged by transplantation of B16F10 tumor cells on alternate flank sites. Delay and do not inhibit tumorsGrowth indicates that in this model, a complete immune memory response cannot be achieved in this single animal.

To assess the effects of oncolytic and immunization on tumor growth, NDV variants R116i and S116 encoding hGM-CF or mGM-CSF, respectively, were tested for efficacy in a mouse syngeneic immunocompetent CT26 colorectal tumor model. Each virus was mixed with 1x10⁸PFU virus was administered intratumorally for 4 doses. Tumors were at least 100mm before dosing began³. All animals treated with the virus demonstrated effective antitumor activity as monotherapy. 11/12 animals were tumor free after treatment with R116 encoding human GM-CSF, which is a 92% complete response rate. The less soluble re-derived S116-KM virus had reduced tumor growth inhibition, achieving 53% TGI and 36% complete response. However, in the presence of murine GM-CSF (which is different from human GM-CSF and will be activated in a mouse model), this response rate increased to 54%, with 75% tumor growth inhibition. Therefore, it is likely that the anti-tumor activity could be improved by equipping 116 virus with GM-CSFS.

Residual tumors were taken for histological analysis and stained by hematoxylin and eosin staining (H and E) and immunohistochemical methods were used for NDV detection (fig. 15C). There is clear evidence that NDV expression, and this appears to be concentrated around the necrotic region of the tumor, suggesting that NDV causes tumor cell death and necrosis. There is also clear evidence of immune cell infiltration into the necrotic area of the tumor. Multinucleated cell syncytia formation was also noted in regions with strong staining for NDV. Furthermore, there appears to be a small residual active tumor, indicating that tumor growth inhibition data may underestimate NDV activity in this model.

Figure 15C shows that NDV has potent anti-tumor activity in an immunocompetent mouse CT26 colorectal tumor model. To assess the effects of oncolytic and immune on tumor growth, NDV encoding hGM-CF or mGM-CSFVariants R116i and S116 were each tested for efficacy in a mouse syngeneic immunocompetent CT26 colorectal tumor model. Each virus was mixed with 1x10⁸PFU virus was administered intratumorally for 4 doses. Tumors were at least 100mm before dosing began³. All animals treated with the virus demonstrated effective antitumor activity as monotherapy. 11/12 animals were tumor free after treatment with R116 encoding human GM-CSF, which is a 92% complete response rate. The less soluble re-derived S116-KM virus had reduced tumor growth inhibition, achieving 53% TGI and 36% complete response. However, in the presence of murine GM-CSF (which is different from human GM-CSF and will be activated in a mouse model), this response rate increased to 54%, with 75% tumor growth inhibition. Thus, the use of GM-CSFS to arm 116 virus can increase anti-tumor activity.

Figures 15D-F show that multiple doses with rNDV R116i caused tumor growth inhibition and driven recruitment of immune cells into ovarian cancer (OVCAR4) xenograft models. To further evaluate the in vivo oncolytic activity of rNDV, a human ovarian cancer xenograft model (OVCAR4) was utilized. This model is growth retarded and has an overall pathology reminiscent of human ovarian tumors, with poorly differentiated cell morphology and extensive ascites-like fluid-filled regions. Once the tumor reaches 100mm³Mice were randomly assigned to receive 8 doses of R116i NDV encoding mouse or human GM-CSF or PBS as a control. In the mouse model, only products derived from the murine gene sequences are biologically active. 2.5x 10⁷pfu was injected into the tumor at a volume of less than 50 μ l once a week. The tumor growth curve is shown in fig. 15D. Long-term tumor suppression is achieved in tumor-bearing animals treated with NDV variants. There was also evidence of viral genome using RT-PCR within the tumor at the end of the study (24 hours after the last dose). Following histological analysis, there was clear evidence of immunoinfiltration in the residual tumor along with other histological changes (fig. 15E and F). The treated tumors were less dedifferentiated than the control treated animals and appeared to have less ascites fluid-filled areas. High found in multiple treated tumors adjacent to the remaining tumorsHorizontal inflammatory infiltrates and Immunohistochemical (IHC) analysis revealed that these immunoinfiltrates were positive for NDV protein. In the NDV treated group, there was a strong recruitment of innate immune cells into the tumor. These data demonstrate potent anti-tumor efficacy in morphologically relevant ovarian cancer models that can be caused by direct oncolytic action and innate immune recruitment and activation.

Example 14: NDV virus-induced tumor regression

73T-R116i-hGM-CSF and 73T-R116i-mGM-CSF were evaluated for oncolytic effects in the B16 melanoma model. The study evaluated viral tolerance in B16 mice. Each virus was expressed at 2x 10⁷pfu was administered intravenously (i.v) or intraperitoneally (i.p) twice on days 11 and 14, or once on day 11 at 1.1x 10⁷pfu was administered intratumorally. The group treated with R116-hGM-SCF or mGM-SCF by three different routes of administration had a slower tumor growth rate compared to the untreated group (FIG. 15A). Each group included 3 mice. The tumor inhibition rate was statistically significant compared to the control group. Thus, the r73T derivatives of the invention have advantageously low avian pathogenicity, high oncolytic activity, and replication to high titers in eggs. Based on these results, the viruses of the present invention can be used to induce tumor regression and improve the therapeutic outcome in cancer patients (fig. 16).

In addition to GM-CSF, multiple transgenes (Table 2) can be inserted into the NDV73T virus strain to enhance tumor killing. These transgenes include the following:

(1) cytokines or engineered variants of cytokines, e.g., GM-CSF, IL-2, IL-21, IL-15, IL-12, and IL-12p70

(2) Cell surface ligands and chemokines, including OX40L, CD40L, ICOSL, Flt3:, B.1(CD80), CD137L, CXCL10(IP-10), CCL5, CXCL 9. (3) Inhibitors of Myc: omomyc. (4) Transgenics for in vivo imaging purposes, such as sodium iodide symporter protein (NIS) -mediated radiotherapy, are used for radiation therapy. (5) Additional modulators that enhance tumor cell survival by tumor killing include, but are not limited to: inhibitors of cell cycle progression, inhibition of anti-apoptotic proteins, enhancement of pro-apoptotic proteins, inhibition of key oncogenic drivers of malignant transformation. These may include transgene delivery of proteins after selective NDV replication in tumor cells, production of selective or broadly active sirnas, delivery of mirnas or inhibition of selected mirnas. (6) Tumor antigens such as E6, E7, cancer testis antigens, carcinoembryonic antigens, artificial or overexpressed proteins, alone or in combination with other transgenes, are novel tumor antigens. (7) Antibodies or recombinant fusion proteins that target immunomodulatory proteins to block negative regulation or provide an agonistic signal to enhance T-cell function. Examples of such antibodies may include, but are not limited to: PD-L1, CTLA4, CD-137(4-1BB), OX40, GITR, TIM-3, CD73, PD-1, HVEM, and LIGHT. (8) Increasing the pharmacodynamic/pharmacokinetic activity of recNDV by engineering or expressing recNDV in cells that transfer proteins to recNDV to reduce clearance by complement or to reduce the adaptive immune response to NDV.

TABLE 5 transgenes possibly inserted into NDV73T and their biological activities

Example 15 cancer therapy involves the administration of oncolytic NDV in combination with an immunomodulatory mAb.

NDV oncolytic viruses may be administered simultaneously or sequentially with a therapeutic antibody or agonist fusion protein where appropriate (e.g., anti-PD-L1, anti-CTLA 4, anti-OX 40, anti-GITR, anti-TIM-3, anti-PD-1, and anti-ICOS). Preclinical data, which forms the most effective dose and schedule of molecules, is generated in combination with the novel NDV constructs described herein to enhance NDV activity in tumor models. Transgenes can be inserted into recombinant NDVs for expression, alone or in combination, to deliver multiple patterns of activity, e.g., to enhance tumor cell death induced by novel variants of NDVs. Increasing the release of tumor cell antigens in combination with immunomodulating approaches has the potential to increase the adaptive immune response to these released tumor antigens.

Example 16 in F protein having R, S or S-KM mutation at the F protein cleavage site, F protein cleavage efficiency and fusion activity were decreased.

To understand whether differences in F protein cleavage sites affect F protein cleavage in infected cells and their effect on fusion activity, F protein plasmids were transfected into 293 cells to examine F protein cleavage. In addition, the F and HN plasmids were co-transfected to examine fusion activity in transition experiments, since both F and HN proteins were required for fusion formation (fig. 3B and 3C). By the host protease Wt F protein being cleaved more efficiently than F proteins with R, S or S-KM mutations at the F protein cleavage site, very little F protein cleavage product (F1) was detected in F with S cleavage site and no cleavage product was detected in cells transfected with the low virulence F plasmid. The F protein of the R and S constructs had n-linked glycosylation sites (NXT) at the cleavage site, resulting in slower mobility on SDS-PAGE than the lento and S-KM, since the KM mutation abolished the glycosylation site. These data demonstrate that R and S mutations at the F protein cleavage site affect F protein cleavage and that S-KN is more efficient than S. Since the F and HN genes were each cloned in pVitro2-neo-MCS vector encoding the EGFP gene, syncytial formation could be visualized by fused green cells. Large syncytia formation was observed in 293 cells transfected with wt F and HN plasmids. All F mutations with R, S or S-KM residues at the F protein cleavage site resulted in greatly reduced syncytia formation.

Example 17R 73T-R116i virus with 198nt insertion exhibited slower growth and differential RNA and protein synthesis profiles in DF-1 cells compared to vilo cells.

Under high moi conditions as shown in fig. 6B and 6D, insertion of 198nt of R116i virus at the HN-L junction exhibited slower growth kinetics in chicken DF-1 cells. The difference in growth of R116i with a 198nt insertion was reduced compared to wt and R viruses, which had no intergenic insertion at the early time point of infection (10-20 hours). In vero cells, the growth differences among these viruses were not very pronounced (fig. 6C and 6E). To understand whether intergenic insertions in the R116i virus affect viral RNA transcription and replication, viral RNA and protein synthesis in infected DF-1 cells were examined by northern blot analysis and immunoblot analysis, respectively (fig. 6F and 6G). RNA and protein synthesis of R116i with a 198nt insertion was greatly reduced in infected DF-1 cells. In contrast, in infected veronol cells, upstream gene transcripts such as NP mRNA were greatly increased in R118i-198 or 198RSV infected cells, whereas the genomic RNA of both viruses was reduced. The levels of L protein were reduced for both viruses, but the other viral protein products were increased, as shown for NP, F and HN in fig. 6H and I. The data obtained in fig. 6F-I were performed under high moi conditions (moi ═ 5). RNA and protein synthesis was further evaluated under low moi conditions (moi ═ 0.001). In addition, R116i-198 or 198RSV exhibited differential RNA and protein synthesis profiles in DF-1 and Verilor cells. RNA and protein synthesis of R116i-198 or 198RSV was reduced in chicken DF-1 cells (FIG. 6J). In contrast, upstream RNA and protein were increased in vilo cells, but levels of L mRNA and L protein were reduced due to the intergenic insertion of the 198nt sequence (fig. 6K). Viral RNA and protein synthesis were further compared in human cell lines, human fibrosarcoma HT1080 and hela cells, and DF-1 cells (fig. 6L). These data confirm that R116i-198 or 198RSV have differential expression, with increased upstream RNA and protein synthesis, whereas L protein expression is down-regulated. These data explain why the R116i-198 or 198RSV viruses replicate well in mammalian cell lines such as Vero, human HT1080, and Hela cells, but do not grow well in chick embryos and chick DF-1 cells. It also provides the basis for the reduced chicken pathogenicity of these R116i viruses, as evidenced by their low intracerebral pathogenicity index (ICPI) values.

Example 18 in R116i-198RSV, mouse GM-CSF transgene expression had lower tumor growth inhibition efficacy than human GM-CSF transgene expression, but not in S116-KM.

FIG. 11C compares the contribution of mGM-CSF to hGM-CSF transgene expression on the oncolytic activity of R116i-198RSV and S116-KM in a HT1080 xenograft mouse tumor model. The hGM-CSF transgene does not cross-react with GM-CSF, and therefore it was used as a control. mGM-CSF transgene in intratumorally administered R116i-198 was less potent in tumor growth inhibition than hGM-CSF. Similar tumor growth inhibition was observed for mGM-CSF and hGM-CSF against S116 virus. Viral titers in virus-treated tumors were determined by plaque assay (fig. 11D). On day 4 post tumor injection, similar titers were detected against R116i-198RSV and S116-KM with mGM-CSF or hGM-CSF pairs. However, on day 7 post-injection, no R116i-198RSV with mGM-CSF was detected, whereas R116i-198RSV with hGM-CSF still had a viral titer of about 4.5logs/g tissue. There was no difference in the virus titer of S116 with hGM-CSF or mGM-CSF. These data indicate that expression of mGM-CSF by R116i contributes to viral clearance.

Immune cell infiltration in virus-infected tumor tissues was examined (fig. 11E). R116i-198RSV with mGM-CSF had the greatest numbers of neutrophils, NK and macrophages in the treated tumors compared to R116-198RSV with hGM-CSF and S116hGM-CSF and with mGM-CSF pair. Cytokines and chemokines were determined in virus-treated tumors by the lewis assay (fig. 11F). All four viruses stimulate cytokine and chemokine production several fold compared to untreated tumor tissue. R116i expressed mGM-CSF approximately 10-fold higher (106.7-and 9.9-fold) than S116.

FIG. 11G shows similar tumor growth inhibitory activity of R116i inserted at 198 or 318nt between HN-L junctions containing the same hGM-CSF transgene in a HT1080 xenograft mouse tumor model. The 318 nucleotide insertion in the HN-L junction region did not reduce viral oncolytic virus activity.

Example 19. evaluation of complement mediated NDV inactivation and regulatory proteins in complement escape role.

The complement (C') system is the primary defense system against microbial infection in the host. There are about 30 different glycoproteins in the human complement system, of which 20 act in plasma and 10 are regulators or receptors on the cell membrane. The membrane-bound C' Regulator (RCA) includes 4 well-characterized molecules: hCD46, hCD55, hCD59, and hCD 35. Their primary function is to protect human cells against autologous complement attack without affecting the role of C' in eliminating exogenous agents. These RCA proteins are host species specific. NDV used in the past for viral therapy was generally produced in chicken embryos. It is expected that NDV oncolytic viruses administered by intravenous injection to cancer patients can be cleared rapidly, thus reducing effective viral administration. Since enveloped viruses produced from human cells incorporate RCA proteins during their egress from infected cells, it is desirable to produce NDV in human cell culture to reduce C' mediated viral lysis or inactivation.

The sensitivity of NDV to C' mediated inactivation was assessed by examining NDV produced in chick embryo, human 293 and hela S3 suspension cell lines (fig. 17). Viruses grown in eggs are sensitive to C' inactivation and serum inhibition is abolished by heat treatment (56 ℃ for 30 minutes) of the serum. Guinea pig complement had similar levels of inactivation of the virus (data not shown), confirming that virus inactivation was due to C'. In addition, viruses produced from hela cells are more resistant to human C' mediated viral inactivation than viruses produced in 293 cells and eggs. Thus, the data indicate that hela cells are better than 293 cells for NDV oncolytic virus production, which may result in lower viral clearance and thus an increased therapeutic index.

To explain why NDV produced from hela S3 cells was more resistant to C', 293 and hela S suspension cell lines were evaluated against the levels of 4 well-characterized human RCA proteins (hCD46, hCD55, hCD59 and hCD 35). hCD35 was not detected in 293 and hela cells by western blot analysis, and thus the data are not shown in figure 18.hCD46、hCD55, and hCD59 were detected in higher abundance in hela S3 cells than in 293 cells. Thus, the level of RCA protein is inversely proportional to the sensitivity of the virus to C'.

To determine whether all three RCA proteins modulate C' function, hCD55, hCD59 orhCD46The transgene is inserted into the NDV genome and a recombinant virus is produced that expresses each of the three RCA proteins. Western blot analysis showed that each of these RCA proteins was expressed by the virus and incorporated into the virion (fig. 19). hCD55 was identified as the major RCA protein conferring C' inactivation function (fig. 20). The hCD 55-expressing virus produced in eggs (hCD55 incorporated into virions) was the most resistant to C' mediated inactivation, in close proximity to the virus produced in hela cells. In contrast, hCD46 has an improvement in marginalization and hCD59 has no detectable effect in C 'regulation for viruses resistant to C' inactivation.

In conclusion, hela cells are considered to be cell lines selected for virus production in order to reduce viral clearance for oncolytic virus therapy and to improve NDV therapeutic index.

The results described herein were obtained using the following materials and methods.

Cells and viruses.

The following cell lines and corresponding media were used: african green monkey kidney vervain cell line (ATCC) and human fibrosarcoma (HT1080, ATCC), eagle's minimal essential medium (EMEM, Hyclone) with 10% Fetal Bovine Serum (FBS); vilo cell clone 51D11 line (medicinal immunization corporation), serum-free medium with 1% glutamine (SFMMegaVir, Hyclone), normal human skin fibroblasts (CCD1122Sk, ATCC), and Iscove's Modified Dulbecco's medium (IMEM) formulated with ATCC with 10% FBS. Recombinant Newcastle Disease Virus (NDV) was grown in allantoic cavity, vilo, or vilo clone 51D11 cells of 10-11 day old Special Pathogen Free (SPF) chick embryos.

Construction of NDV antigenomic cDNA and supporting plasmid NP, P and L.

The viral RNA of NDV strain 73T was obtained from doctor marke pierce (MarkPeeples) national Children's Hospital (national world Children's Hospital). NDV sequences (GenBank) were aligned to obtain a consensus sequence to design DNA oligonucleotides for RT-PCR of viral RNA. Six sub-genomic cDNA overlapping fragments spanning the entire NDV genome were generated by high fidelity RT-PCR (FIG. 1). The pUC19 vector was modified to include an 88nt oligonucleotide linker containing restriction sites introduced between the EcoRI and HindIII sites for the sequential assembly of the full-length antigenomic cDNA of the NDV73T strain. In addition, the 73T strain cDNA plasmid (p73T) contained a 27 nucleotide (nt) T7RNA polymerase promoter at the 5 'end, 189 nt containing the HDV antigenomic ribozyme sequence and a T7RNA polymerase transcription termination signal at the 3' end. To generate non-virulent NDV, the sequence encoding the protease cleavage site of the fusion protein is modified by site-directed mutagenesis to those of the (lentogenic) or cytomegalovirus glycoprotein b (gb) of the non-virulent NDV LaSota strain (S116). For construction of NP, P and L expression plasmids, the protein Open Reading Frame (ORF) was amplified by RT-PCR and cloned into plasmid pCITE2a under the control of T7RNA polymerase.

The transgene is inserted into NDV.

For the insertion of the transgene at the P-M junction, an AfeI restriction site was introduced at nt3148 in the subcloning plasmid containing the SacII-PmlI fragment (FIG. 2A). cDNAs encoding human or mouse granulocyte macrophage colony stimulating factor (GM-CSF) or interleukin 2(IL-2) were codon optimized and synthesized by DNA 2.0. The gene cassette, which contains the gene end of N (GE), the gene start of P (GS) and the open reading frame of the transgene (ORF), is inserted into the AfeI site. The SacII-PmlI fragment from the resulting plasmid was transferred into the plasmid r73T and designated P73T-P1.

To insert the transgene at the HN-L junction between the HN ORF and the gene end signal (GE) sequence of HN, an AfeI restriction site was introduced at nt 8231 in the plasmid containing the AgeI-XbaI fragment (FIG. 2A). The gene cassette was generated by PCR using a pair of phosphate sense and antisense primers (table 3) and inserted into the AfeI site.

TABLE 3 oligonucleotide primer sequences for insertion of transgenic transcription cassettes.

The Gene End (GE) and Gene Start (GS) sequences are underlined. Kozak sequence is shown in lower case letters. Sequences corresponding to the 5 'or 3' sequences of the transgene are shown in italics. In addition to EGFP (H-N), all other primer pairs can be used to insert transgenes between G-M or HN-L.

hGM-CSF: human granulocyte macrophage colony stimulating factor; mGM-CSF: mouse GM-CSF; hIL-2 and mIL-2, corresponding to human and mouse interleukin 2(IL-2), respectively.

The AgI-XbaI fragment from the resulting plasmid was transferred to plasmid p73T, yielding p73T-HN 1. Another strategy to insert sequences at the HN-L junction is to insert transgene cassettes or sequences from other paramyxoviruses between the gene end signal (GE) of HN and the gene start signal (GS) of L (fig. 4) at the AfeI site, which is introduced at nt 8359. The FL cDNA plasmid was designated p73T-R116 i. Since the NDV genome length must be in multiples of 6 nucleotides (6 rules), the antigenomic cdnas of the various constructs are made to follow 6 rules.

To insert the two transcription cassettes into the P-M junction, an AfeI site was introduced at the end of the ORF of GM-CSF (nt 3619) (FIG. 2B). The IL-2ORF was amplified using a pair of phosphate sense and antisense primers containing GE and GS sequences and inserted at the AfeI site. The SacII-PmlI fragment from the resulting plasmid, including the GM-CSF and IL-2 transcription cassettes, was replaced back into plasmid r73T, yielding P73T-P2.

R73T chimeric viruses were generated that contained the extracellular domains of other paramyxoviruses.

Chimeric NDV genomic DNA was generated by replacing F and HN of NDV with those of pigeon paramyxovirus type 1 (PPMV-1). The C-terminal coding sequence for the cytoplasmic tail and transmembrane portions (amino acid residues 503 to 553) of NDV73T F was ligated to the extracellular domain F protein coding sequence of PPMV-1 (residues 1 to 502), and the N-terminal coding sequence of NDV HN (amino acid sequence residues 1 to 45) was fused to HN (residues 46 to 577) by an overlap PCR reaction using the GeneArt kit (Invitrogen). The amplified fragment was digested and cloned into PmlI-AgeI digested NDV cDNA. Parainfluenza virus 5(PIV-5) F or HN was introduced into the NDV73T antigenomic cDNA by a similar cloning strategy. The PIV5F (residues 1 to 486) extracellular domain was fused to the transmembrane and cytoplasmic tail of NDV73T F (residues 503 to 553). NDVHN (residues 1 to 45) was linked to PIV5HN extracellular domain (residues 36 to 565). The cDNA fragment was cloned into PmlI-AgeI digested NDV antigenomic cDNA.

Recovering the recombinant NDV from the transfected cDNA plasmid.

A mammalian cell line expressing T7RNA polymerase, such as BHK-T7 cells, was transfected with plasmids expressing NDVNP, P, and L proteins (0.4. mu.g, and 0.2. mu.g/well in 6-well plates, respectively) and a plasmid encoding NDV antigenomic cDNA (1.6. mu.g) using Lipofectamine 2000. Three days after transfection, cell culture supernatants were injected into the allantoic cavities of 10 to 11-day-old SPF chick embryos and passaged in Verilor cells to amplify the rescued virus. Recovery of virus was confirmed by hemagglutination assay using 1% chicken Red Blood Cells (RBC). Rescue of viruses can also be performed by electroporation of NP, P, L, antigenomic cDNA plasmids into vero cells along with plasmids expressing T7RNA polymerase as described previously (coul (Kaur) et al, Optimization of plasmids-for human trials, rescue of highly attenuated and thermosensitive Respiratory Syncytial Virus (RSV) vaccine selection only (RSV) vaccine 2008, journal of virology methods (j.v. methods)153: 196-. The recovered virus was confirmed by sequencing the RT-PCR amplified cDNA.

In vitro passaging was used to select viruses with stable F protein cleavage sites.

To test if the F Protein Cleavage Sequence (FPCS)) is stable and any stabilized mutations can be selected after passage in tissue culture, r73T-S116 was serially passaged for 10 times in vilo and human fibrosarcoma HT1080 cells at an MOI of 0.01. After every 2-3 passages, viral RNA was isolated from the medium, cDNA was amplified by RT-PCR, and the F and/or HN genes were sequenced.

Viral plaque morphology and titer quantification by plaque assay in vero cells.

Villolo cells on 6-well plates were infected with serial dilutions of the virus and incubated at 37 ℃ for plaque morphology for 3 or 6 days with 1% methylcellulose coverage, in trypsin (TrpyLE)^TMInvitrogen) for virus titerAnd (4) transforming. Cell monolayers were fixed with methanol and stained with chicken anti-NDV polyclonal antibody against fully inactivated NDV virus, followed by exposure to horseradish peroxidase (HRP) -conjugated anti-chicken antibody (Dako).

Viral chicken pathogenicity test as determined by mean egg death time (MDT) and intracerebral pathogenicity index (ICPI).

The pathogenicity of the r73T virus was determined by mean time to death (MDT) testing in 10 day old SPF chick embryos. ICPI testing in SPF chickens at 1 day of age was performed at the national veterinary services laboratory of the united states department of agriculture (NVSL, emms, iowa). For the MDT test, this will be at 10^-6And 10^-90.1ml per dilution in a series of 10-fold dilutions in between were inoculated into allantoic cavities of 8-10 9-10 day-old eggs and incubated at 37 ℃. Eggs were examined twice daily for 7 days to record embryo death time. The MDT was calculated as the average time (hours) that the minimum lethal dose of virus killed all inoculated embryos. MDT assays provide a reasonable prediction of viral pathogenicity. Having MDT<The 60 hour virus is typically a highly virulent (virulent) strain; in the case of MDT 60 to 90 hours, is a mesogenic (intermediate) virus strain;>as a low virulence (non-virulent) virus strain for 90 hours. For the ICPI test, 0.05ml of 1:10 diluted freshly infected allantoic fluid per virus was inoculated into a panel of 10 1-day-old SPF chickens by an intracerebral route. They were observed every 8 hours for clinical symptoms and mortality for a period of 8 days. At each observation, they were classified as follows: if normal 0, if sick 1, and if dead 2. ICPI is the average score of each observation over an 8 day period. ICPI values range from 0.0 to 2.0. Attenuated toxicity (LoND): ICPI<0.7; virulent toxin (vND): ICPI is more than or equal to 0.7.

Viral cell killing assessed by cell viability assay.

Cells were treated at 5X10³Cells/well were placed in 96-well plates and infected with r73T overnight at different MOIs. Cell viability was determined by CellTiter Glo kit (plomega) according to manufacturer's manual. By comparing the ATP level of each test sample to 100% viable untreated samplesThe comparison determines the relative percentage of surviving cells. The data presented in the table are relative percentages of cells killed.

NDV tumor killing assessed in a subcutaneous HT1080 xenograft model.

Athymic NCR syngeneic nude mice (Taconc) were treated with 5X10⁶Ht1080 cells (in 100 μ L PBS) were implanted subcutaneously (s.c.) into one flank. When the tumor reaches 65-300mm³At volume (c), viral therapy is initiated. Recombinant 73T in 100 μ l was administered locally by intratumoral (i.t) injection or systemically by intratumoral (i.t) injection into the tail vein at different dose levels, respectively. Control animals were injected with 100 μ L PBS only. Tumor growth was measured using digital calipers and tumor volume was calculated as 0.5x (height) x width x length (mm)³). When the body weight is reduced by 20% of the original body weight or the tumor volume exceeds 2000mm³At that time, the mice were sacrificed.

Viral biodistribution in Ht1080 xenografted mice.

Nine nude mice bearing HT1080 human fibrosarcoma xenograft subcutaneous tumor were used 10⁸pfu R73T-R116i-hGM-CSF for i.v injection. Three mice were terminated on days 1, 4, and 8 post injection. One mouse injected with PBS was terminated on day 8. Tumor, lung, spleen, ovary and serum samples were collected. Infectious virus titers in tissue homogenates were quantified by plaque assay.

GM-CSF protein levels were quantified by ELISA.

Tumors from NDV-infected and PBS-injected mice were homogenized in PBS using a MACS separator (Miltenyi Biotec, american and whirlwind biotechnology, germany) according to the manufacturer's instructions. Supernatants from homogenized tissues and sera collected from mice were tested for levels of GM-CSF by the Duoset ELISA kit (Andy Bio (R & D)).

Statistical analysis

All statistical analyses were performed using GraphPad Prism 6.0 software. Unpaired t-tests were used to assess differences in tumor regression between groups. IC50 for rNDV 73T was also calculated for in vitro cell killing in normal as well as tumor cells using GraphPadPrism software.

Other embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adapt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

Reference to a list of elements in any definition of a variable includes herein the definition of the variable as any single element or combination (or subcombination) of the listed elements. The detailed description of embodiments herein includes embodiments as any single embodiment or in combination with any other embodiments or portions thereof.

All patents, publications, CAS, and accession numbers mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent, publication, or accession number was specifically and individually indicated to be incorporated by reference.

Claims

1. An attenuated Newcastle Disease Virus (NDV) comprising an F protein cleavage site (S116) derived from glycoprotein b (gb) of Cytomegalovirus (CMV), wherein the modified F Protein Cleavage Sequence (FPCS) comprises a sequence selected from the group consisting of:

-S116：¹¹¹H-N-R-T-K-S/F¹¹⁷

-S116K：¹¹¹H-N-K-T-K-S/F¹¹⁷

-S116M：¹¹¹H-N-R-M-K-S/F¹¹⁷

-S116KM：¹¹¹H-N-K-M-K-S/F-I¹¹⁸

-R116：¹¹¹H-N-R-T-K-R/F-I¹¹⁸。

2. the attenuated newcastle disease virus of claim 1, wherein the attenuated virus strain is a modified 73T virus strain.

3. The attenuated newcastle disease virus of claim 1, wherein the attenuated NDV virus is a R73T-R116 virus.

4. The attenuated newcastle disease virus of claim 1, wherein the virus comprises an increased HN-L intergenic region.

5. The attenuated newcastle disease virus of claim 4, wherein the HN-L intergenic region comprises a non-coding sequence of between at least about 50-300 nucleotides in length.

6. The attenuated newcastle disease virus of claim 5, wherein the non-coding sequence is derived from paramyxovirus type 1 (APMV-1), Respiratory Syncytial Virus (RSV), or a random sequence.

7. The attenuated newcastle disease virus of claim 5, wherein the non-coding sequence between the HN and L genes is 60nt, 102nt, 144nt, 198nt, or 318nt in length.

8. The attenuated newcastle disease virus of any of claims 1-7, wherein the virus comprises one or more heterologous polynucleotide sequences inserted at the P-M junction and/or HN-L junction.

9. The attenuated newcastle disease virus of claim 8, wherein the virus comprises two or more heterologous polynucleotide sequences, wherein at least one heterologous polynucleotide sequence is inserted at the P-M junction and at least one is inserted at the HN-L junction.

10. The attenuated newcastle disease virus of claim 8, wherein the heterologous polynucleotide sequence is a transgene encoding a polypeptide that enhances the oncolytic properties of the virus.

11. The attenuated newcastle disease virus of claim 9, wherein the transgene encodes a cytokine, cell surface ligand, and/or chemokine.

12. The attenuated newcastle disease virus of claim 11, wherein the cytokine is selected from the group consisting of: GM-CSF, IL-2, IL-21, IL-15, IL-12, and IL-12p 70.

13. The attenuated newcastle disease virus of claim 12, wherein the cytokine is human GM-CSF.

14. The attenuated newcastle disease virus of claim 8, wherein the heterologous polynucleotide sequence is a transgene encoding a detectable moiety.

15. The attenuated newcastle disease virus of claim 14, wherein the expression level of the detectable moiety is correlated with viral replication.

16. The attenuated newcastle disease virus of any of claims 1-7, wherein the F and HN genes of NDV are replaced with the corresponding extracellular domains of canine parainfluenza virus 5(PIV 5) or pigeon paramyxovirus type 1 (PPMV-1).

17. The attenuated newcastle disease virus of claim 1, wherein the virus is 73T-R116 i-hGM-CSF.

18. The attenuated newcastle disease virus of claim 1, wherein the attenuated virus has a mean time to death (MDT) in ovo of greater than 90 hours or about 90-156 hours.

19. The attenuated newcastle disease virus of claim 1, wherein the attenuated virus has an intracerebral pathogenicity index of between about 0-0.7.

20. The attenuated newcastle disease virus of claim 1, wherein the attenuated virus has an intracerebral pathogenicity index of about 0.

21. The attenuated newcastle disease virus of claim 1, wherein the attenuated virus has less than about 15% cytotoxicity in HT1080 cells.

22. The attenuated newcastle disease virus of claim 1, wherein the attenuated virus selectively kills tumor cells with a killing efficiency of at least 10% or 15%.

23. The attenuated newcastle disease virus of claim 22, wherein the tumor cell killing efficiency is between about 75-100%.

24. Use of an attenuated newcastle disease virus according to any of claims 1-23 for the preparation of a pharmaceutical composition for use in a method of selectively killing a tumor cell, which method comprises contacting a tumor cell with an attenuated newcastle disease virus according to any of claims 1-23.

25. Use of the attenuated newcastle disease virus of any of claims 1-23 in the preparation of a pharmaceutical composition for use in a method of inducing tumor cell regression in an individual, the method comprising contacting a tumor cell with the attenuated newcastle disease virus of any of claims 1-23.

26. Use of an attenuated newcastle disease virus according to any of claims 1-23 for the preparation of a pharmaceutical composition for use in a method of reducing tumor cell survival or proliferation, the method comprising contacting a tumor cell with an attenuated newcastle disease virus according to any of claims 1-23.

27. The use of any one of claims 24-26, wherein the cell is a cancer cell selected from the group consisting of: bladder cancer cells, ovarian cancer cells, brain cancer cells, pancreatic cancer cells, prostate cancer cells, sarcoma cancer cells, lung cancer cells, breast cancer cells, cervical cancer cells, bone cancer cells, liver cancer cells, head and neck cancer cells, stomach cancer cells, kidney cancer cells, melanoma cancer cells, lymphoma cancer cells, leukemia cancer cells, thyroid cancer cells, colon cancer cells, colorectal cancer cells, and melanoma cancer cells.

28. Use of the attenuated newcastle disease virus of any of claims 1-23 in the preparation of a pharmaceutical composition for use in a method of treating neoplasia in an individual, the method comprising administering to the individual an effective amount of the attenuated newcastle disease virus of any of claims 1-23.

29. The use of any one of claims 24-26 and 28, wherein the attenuated newcastle disease virus is r 73T-S116.

30. The use of claim 25, wherein the attenuated newcastle disease virus is delivered systemically, intraperitoneally, or intratumorally.

31. The use of claim 25, wherein the virus is administered at about 10%⁷pfu to about 10⁹Doses of pfu were administered.

32. The use of claim 25, wherein the virus is administered at about 10%⁹pfu to about 10¹¹Doses of pfu are administered intravenously.

33. The use of claim 28, wherein the individual has a neoplasia selected from the group consisting of: bladder cancer, ovarian cancer, brain cancer, pancreatic cancer, prostate cancer, sarcoma, lung cancer, breast cancer, cervical cancer, bone cancer, liver cancer, head and neck cancer, gastric cancer, renal cancer, melanoma, lymphoma, leukemia, thyroid cancer, colon cancer, colorectal cancer, and melanoma.

34. Use of the attenuated newcastle disease virus of any of claims 1-23 in the preparation of a pharmaceutical composition for use in a method of treating neoplasia in an individual who has developed an immune response against NDV, the method comprising administering an effective amount of the attenuated chimeric newcastle disease virus of any of claims 1-23, wherein the virus is a chimeric virus comprising F and/or HN genes of canine parainfluenza virus 5(PIV 5) or pigeon paramyxovirus type 1 (PPMV-1), wherein said chimeric newcastle disease virus is antigenically different from NDV.

35. The use of claim 28, wherein the method increases the level of oncolytic virus present in the individual relative to the level of oncolytic virus present in a control individual that has developed an immune response against NDV but has not received the chimeric newcastle disease virus.

36. A nucleic acid comprising a full-length cDNA of 73T, wherein the nucleic acid encodes a modified F protein cleavage sequence selected from the group consisting of:

-S116：¹¹¹H-N-R-T-K-S/F¹¹⁷

-S116K：¹¹¹H-N-K-T-K-S/F¹¹⁷

-S116M：¹¹¹H-N-R-M-K-S/F¹¹⁷

-S116KM：¹¹¹H-N-K-M-K-S/F-I¹¹⁸

-R116：¹¹¹H-N-R-T-K-R/F-I¹¹⁸。

37. a vector comprising a full-length cDNA of 73T, wherein the vector encodes a modified F protein cleavage sequence selected from the group consisting of:

-S116：¹¹¹H-N-R-T-K-S/F¹¹⁷

-S116K：¹¹¹H-N-K-T-K-S/F¹¹⁷

-S116M：¹¹¹H-N-R-M-K-S/F¹¹⁷

-S116KM：¹¹¹H-N-K-M-K-S/F-I¹¹⁸

-R116：¹¹¹H-N-R-T-K-R/F-I¹¹⁸。

38. a virulent particle comprising the nucleic acid of claim 36.

39. A host cell infected with the attenuated newcastle disease virus of any of claims 1-23.

40. A host cell comprising the nucleic acid of claim 36.

41. The attenuated newcastle disease virus of claim 10, wherein the transgene is selected from the transgenes listed in table 5.